CN108865972A - A kind of cell culture solution - Google Patents

Abstract

The present invention relates to a cell culture solution comprising an inhibitor of bone morphogenetic protein, an inhibitor of glycogen synthase kinase-3β, an agent that binds to leucine-rich repeat-containing G protein-coupled receptor 5, and histone Deacetylase inhibitors.

Description

a cell culture solution

This application is a divisional application. The international application number of the original application is PCT/US2014/023197, the Chinese national application number is 201480027151.4, and the application date is March 11, 2014. The title of the invention is "Used for the expansion and cultivation of epithelial stem cells compositions and methods".

Cross References to Related Applications

This application claims the benefit under 35 U.S.C. §119(e) of US Provisional Application No. 61/783,245, filed March 14, 2013, the entire contents of which are hereby incorporated by reference.

Government funding

This study was supported by National Academy of Dental and Craniofacial Research grant DE013023. The U.S. Government has certain rights in this invention.

Background technique

A monolayer of epithelial cells that actively self-renews and is organized into crypts (Crypts) and villi covers the small intestine. It was recently shown that intestinal epithelial renewal is driven by Lgr5 ⁺ intestinal stem cells (ISCs) residing at the base of these crypts (Barker et al., 2007). Lgr5 ⁺ stem cells can be isolated and cultured in vitro to form organoids containing crypt-villus structures that recapitulate native intestinal epithelium (Sato et al., 2009). Although these stem cells can be expanded into multiple passages in the form of organoids, existing culture conditions offer little or no control over self-renewal and differentiation. A typical culture consists of a heterogeneous population of cells including stem cells and differentiated cells (Sato et al., 2009). In particular, self-renewal and proliferation of Lgr5 ⁺ stem cells both in vitro and in vivo depend on direct cell contact between Lgr5 ⁺ stem cells and another crypt cell type called paneth cells, which allows controlled culture The ability to determine the fate of Lgr5 ⁺ stem cells in organisms is significantly complicated and restricted. The inability to efficiently expand Lgr5 ⁺ stem cells greatly limits the translation of this biology into therapy, where syngeneic stem cell culture and an efficient scale-up process are critical prior to transplantation. Furthermore, there remains a need to develop improved clinically directed systems for ex vivo transplantation of expanded epithelial tissue into damaged recipient organs.

Contents of the invention

In one aspect, the invention provides a cell culture solution.

In one embodiment, the present invention provides an inhibitor comprising an inhibitor of bone morphogenetic protein, an inhibitor of glycogen synthase kinase-3β, an agent that binds to leucine-rich repeat G protein-coupled receptor 5, and a histone Cell culture solution for deacetylase inhibitors. In one embodiment, the inhibitor of glycogen synthase kinase-3β may be CHIR99021, the reagent that binds to leucine-rich repeat G protein-coupled receptor 5 may be R-spondin 1, and the The HDAC inhibitor may be valproic acid.

In another embodiment, the invention provides a cell culture solution comprising an inhibitor of bone morphogenetic protein, at least about 3 [mu]M of CHIR99021, and a histone deacetylase inhibitor.

In yet another embodiment, the present invention provides a cell culture solution comprising an inhibitor of bone morphogenetic protein, an agent that binds to leucine-rich repeat G protein-coupled receptor 5, a Wnt agonist, and an HDAC6 inhibitor.

In yet another embodiment, the present invention provides a cell culture solution comprising an inhibitor of bone morphogenetic protein, R-spondin1, lithium chloride, and a histone deacetylase inhibitor.

In another embodiment, the histone deacetylase inhibitor provided by the invention is a Pan-HDAC inhibitor. The Pan-HDAC inhibitor may be selected from the group consisting of valproic acid, trichostatin A, suberoyl anilide hydroxamic acid and suberoyl hydroxamic acid (SBHA).

In yet another embodiment, the histone deacetylase inhibitor provided by the invention is an HDAC6 inhibitor. The HDAC6 inhibitor can be selected from the group consisting of tubastatin, tubastatin A and compound 7.

In another embodiment, the present invention provides an inhibitor of bone morphogenetic protein which may be selected from the group consisting of noggin, chordrin, follistatin, DAN, DAN-containing cysteine knot structure domain protein, sclerostin, Twisted Gastrulation, uterine sensitivity-related gene-1, connective tissue growth factor, inhibin, BMP-3 and Dorsomorphin.

In yet another embodiment, the present invention provides an inhibitor of glycogen synthase kinase-3β which may be selected from the group consisting of: CHIR99021, LiCl, BIO-acetone oxime, CHIR98014, SB 216763, SB 415286, 3F8, Kenpaullone (Kenpaullone), 1-Azakenpaullone (1-Azakenpaullone), TC-G 24, TCS 2002, AR-A 014418, TCS 21311, TWS 119, BIO-acetone oxime, 10Z-haimendi Plug, GSK-3β Inhibitor II, GSK-3β Inhibitor I, GSK-3β Inhibitor XXVII, GSK-3β Inhibitor XXVI, FRATtide Peptide, Cdk1/5 Inhibitor, and Bikinin.

In another embodiment, the present invention provides a reagent that binds to leucine-rich repeat G protein-coupled receptor 5, which can be selected from R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin A group consisting of 4 spondin.

In yet another embodiment, the present invention provides a Wnt agonist that can be selected from the group consisting of: Wnt-1/Int-1, Wnt-2/Irp (Int-I-related protein), Wnt-2b/ 13. Wnt-3/Int-4, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a/8d, Wnt-8b, Wnt- 9a/14, Wnt-9b/14b/15, Wnt-10a, Wnt-10b/12, Wnt-11, Wnt-16, R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, Norrin, CHIR99021, LiCl, BIO ((2'Z,3'E)-6-bromoisatin-3'-oxime), CHIR98014, SB 216763, SB 415286, 3F8, Campalone , 1-Azacamparone, TC-G24, TCS 2002, AR-A 014418, 2-amino4-[3,4-(methylenedioxy)benzyl-amino]-6-(3- Methoxyphenyl)pyrimidine, IQ 1, DCA, QS 11, WAY-316606, (hetero)arylpyrimidine, 10Z-hemandex, TCS 21311, TWS 119, GSK-3 Inhibitor IX, GSK-3 Inhibitor Agent IV, GSK-3β Inhibitor II, GSK-3β Inhibitor I, GSK-3β Inhibitor XXVII, GSK-3β Inhibitor XXVI, FRATtide, Cdk1/5 Inhibitor, Bikinin and 1-Azacamparone.

In yet another embodiment, the present invention provides a cell culture solution comprising Noggin, R-spondin 1, CHIR99021 and an Atoh1 inhibitor. The Atoh1 inhibitor can be an inhibitory nucleic acid.

In another embodiment, the present invention provides a cell culture solution further comprising epidermal growth factor and/or a Notch agonist. The Notch agonist may be selected from the group consisting of Notch1 antibody (N1 Ab), Delta 1, Delta-like 3, Delta-like 4, Jagged 1, Jagged 2, DSL peptide and Delta D.

In yet another embodiment, the present invention provides about 5 ng/ml to about 500 ng/ml of EGF, about 5 ng/ml to about 500 ng/ml of noggin, about 50 ng/ml to about 1000 ng/ml of R-spondin , CHIR99021 from about 0.1 μM to about 10 μM, and valproic acid from about 0.1 mM to about 5 mM.

In another aspect, the invention provides a cell culture system comprising the cell culture solution of the invention.

In one embodiment, the invention provides a kind of cell culture system, it comprises:

i) epithelial stem cells or epithelial progenitor cells, or a population of epithelial stem cells or epithelial progenitor cells;

ii) R-spondin 1;

iii) CHIR99021;

iv) histone deacetylase inhibitors; and

v) Optional inhibitors of bone morphogenetic proteins.

In another embodiment, the invention provides a kind of cell culture system, it comprises:

i) epithelial stem cells or epithelial progenitor cells, or a population of epithelial stem cells or epithelial progenitor cells;

ii) R-spondin 1;

iii) CHIR99021;

iv) Atoh1 inhibitors; and

v) Optional inhibitors of bone morphogenetic proteins.

In another embodiment, the invention provides a kind of cell culture system, it comprises:

i) epithelial stem cells or epithelial progenitor cells, or a population of epithelial stem cells or epithelial progenitor cells;

ii) R-spondin 1;

iii) lithium chloride;

iv) histone deacetylase inhibitors; and

v) Optional inhibitors of bone morphogenetic proteins.

In another embodiment, the invention provides a kind of cell culture system, it comprises:

i) epithelial stem cells or epithelial progenitor cells, or a population of epithelial stem cells or epithelial progenitor cells;

ii) R-spondin 1;

iii) Wnt agonists;

iv) HDAC6 inhibitors; and

v) Optional inhibitors of bone morphogenetic proteins.

In another embodiment, the cell culture system of the invention comprises epithelial stem cells and populations of epithelial stem cells that may comprise LGR5 positive stem cells.

In another embodiment, the population of epithelial stem cells or epithelial progenitor cells in the cell culture system of the invention comprises at least 30%, 85%, 90, 95%, or 99% of the cells in the system.

In another embodiment, the invention provides a kind of cell culture system, it comprises:

i) Tumor organoids;

ii) a reagent that binds to leucine-rich repeat G protein-coupled receptor 5;

iii) Wnt agonists;

iv) a histone deacetylase inhibitor or an Atoh1 inhibitor; and

v) Optional inhibitors of bone morphogenetic proteins.

In another embodiment, the present invention provides a substrate comprising a submucosa, a coating comprising collagen, and a cell layer comprising any member of the group consisting of epithelial stem cells, isolated tissues comprising epithelial stem cells, and/or epithelial organoids Cell culture system. The collagen-comprising coating can be over or surround the epithelial stem cells, isolated tissue comprising epithelial stem cells, or epithelial organoids. The collagen-containing coating can also be between the SIS substrate and the epithelial stem cells, isolated tissue comprising epithelial stem cells, or epithelial organoids. The submucosa substrate may comprise SIS, and may further comprise epidermal growth factor, bone morphogenetic protein, an agent that binds to leucine-rich repeat G protein-coupled receptor 5, a Wnt agonist, Y-27632, and histone Deacetylase inhibitors. The cell culture system may further comprise the cell culture solution of the present invention, including an inhibitor of bone morphogenetic protein, a reagent that binds to leucine-rich repeat G protein-coupled receptor 5, a Wnt agonist, Y-27632 and a solution of histone deacetylase inhibitors.

In another embodiment, the present invention provides a cell culture system comprising a submucosa substrate comprising epidermal growth factor, bone morphogenetic protein, R-spondin 1, CHIR99021, Y-27632 and histone deacetylase inhibitors. The cell culture system may further comprise a solution comprising epidermal growth factor, an inhibitor of bone morphogenetic protein, R-spondin 1, CHIR99021, Y-27632, and a histone deacetylase inhibitor.

In another aspect, the invention provides methods of forming epithelial organoids from isolated epithelial stem cells.

In one embodiment, the present invention provides a method for efficiently forming epithelial organoids from isolated epithelial stem cells, the method comprising the steps of:

i) incubating the isolated epithelial stem cells in the presence of noggin, R-Spondin 1, CHIR99021 and histone deacetylase inhibitors; and

ii) forming epithelial organoids from said isolated epithelial stem cells, wherein at least about 25%, 40%, 50%, 75%, 90% of said isolated epithelial stem cells form epithelial organoids.

In another aspect, the present invention provides a method for efficiently forming epithelial organoids from a single isolated epithelial stem cell, said method comprising the steps of:

i) incubating said single isolated epithelial stem cells in the presence of noggin, R-Spondin 1, CHIR99021 and a histone deacetylase inhibitor; and

ii) forming epithelial organoids from said isolated epithelial stem cells, wherein at least about 6% of said single isolated epithelial stem cells form epithelial organoids.

In another aspect, the invention provides a method of determining the efficacy of a chemotherapeutic agent relative to a tumor organoid, said method comprising the steps of:

i) incubating the tumor organoid in the presence of an inhibitor of bone morphogenetic protein, R-spondin 1, a Wnt agonist, a histone deacetylase inhibitor, and a chemotherapeutic agent; and

ii) determining a parameter selected from the group consisting of inhibition of cell viability, inhibition of cell proliferation, inhibition of tumor-associated gene expression, activation of apoptosis, and inhibition of cell survival,

Wherein, detecting an increase in said parameter indicates the efficacy of said chemotherapeutic agent relative to a tumor organoid.

In another aspect, the present invention provides a method of forming Paneth cells in a cell culture system, the method comprising incubating epithelial stem cells in the presence of at least one Wnt agonist and at least one Notch inhibitor, wherein The respective amounts of the at least one Wnt agonist and the at least one Notch inhibitor are amounts sufficient to generate Paneth cells.

In one embodiment, said epithelial stem cells may be further incubated in the presence of at least one inhibitor of bone morphogenetic protein.

In another embodiment, the Notch inhibitor is DAPT.

In another embodiment, the epithelial stem cells are LGR5 positive stem cells.

In another aspect, the present invention provides a method of forming intestinal epithelial cells in a cell culture system, the method comprising incubating in the presence of at least one Wnt inhibitor and at least one histone deacetylase inhibitor The epithelial stem cells, the at least one Wnt inhibitor and the at least one histone deacetylase inhibitor are each in amounts sufficient to generate intestinal epithelial cells. The epithelial stem cells may be further incubated in the presence of an inhibitor of epidermal growth factor and/or bone morphogenetic protein.

In one embodiment, the Wnt inhibitor can be selected from IWP-2, XAV-939, ICG-001, LGK-974, IWR-1-endo, KY02111, Wnt-C59, DKK-1, FH-535, Panel consisting of Box5, peptide Pen-N3, anti-SFRP antibody and anti-LRP6 antibody.

In another aspect, the present invention provides a method of forming goblet cells in a cell culture system, the method comprising incubating epithelial stem cells in the presence of at least one Wnt inhibitor and at least one Notch inhibitor, said The respective amounts of at least one Wnt inhibitor and at least one Notch inhibitor are amounts sufficient to generate goblet cells. The epidermal stem cells may be further incubated in the presence of epidermal growth factor.

In one embodiment, the Notch inhibitor can be selected from DAPT, RO4929097, LY450139, LY900009, LY3039478, LY411575, YO-01027, BMS-708163, BMS-906024, compound E, BMS-299897, SAHM1, selective Abeta42 (Abeta42-Selective) and SB 225002 group.

In another aspect, the present invention provides a method of forming enteroendocrine cells in a cell culture system, the method comprising at least one Notch inhibitor and inhibiting the receptor tyrosine kinase, mitogen-activated protein (MAP) Epithelial stem cells are incubated in the presence of at least one agent of kinase or extracellular signal-regulated kinase (ERK), said at least one Notch inhibitor and said agent are each in an amount sufficient to produce enteroendocrine in a cell culture system amount of cells. The epidermal stem cells may be further incubated in the presence of epidermal growth factor, an agent that binds to leucine-rich repeat G protein-coupled receptor 5, and/or an inhibitor of bone morphogenetic protein. The MAP kinase may be a mitogen-activated protein kinase kinase.

In one embodiment, the agent that inhibits MAP kinase may be selected from the group consisting of AS-703026, PD0325901, PD98059, Selumetinib, SL-327, U0126, TAK-733 and Trametinib .

In another embodiment, the agent that inhibits RTK can be selected from Gefitinib (Gefitinib), AG 99, Erlotinib (Erlotinib), Afatinib (Afatinib), Lapatinib (Lapatinib), WZ4002 and AG-18 group.

In another embodiment, the agent that inhibits ERK may be AS-703026 or PD0325901.

In another aspect, the present invention provides a method of forming intestinal epithelial cells in a subject in need thereof, the method comprising administering to said subject a Wnt agonist in an amount sufficient to form intestinal epithelial cells in the subject. agents and histone deacetylase inhibitors. The Wnt agonist may be CHIR99021, and the histone deacetylase inhibitor may be valproic acid. CHIR99021 can be administered in an amount of about 0.1 mg/kg/day to about 100 mg/kg/day, and valproic acid can be administered in an amount of about 1 mg/kg/day to about 1000 mg/kg/day.

In another aspect, the present invention provides a method of forming intestinal epithelial cells in a subject in need thereof, the method comprising administering Wnt to said subject in an amount sufficient to form intestinal epithelial cells in said subject. Agonists and Notch agonists.

In yet another aspect, the invention provides a method of treating a small bowel disorder comprising administering to a subject a Wnt agonist and a histone deacetylase inhibitor or a Wnt agonist and a Notch agonist. In certain embodiments, the small bowel disorder is selected from the group consisting of: enterocolitis; viral infection, such as nonspecific enteritis or specific viral enteritis; diverticulitis; bacterial enterocolitis, such as salmonellosis, Shigellosis, Campylobacter enterocolitis, or Yersinia enterocolitis; protozoan infections such as amoebiasis; helminth infections; and pseudomembranous colitis and pulmonary complications of cystic fibrosis and chronic obstructive pulmonary disease; appendicitis; atrophic gastritis; Barrett's esophagus; pneumonia; cervicitis; chronic interstitial nephritis; colitis; colonic diverticulitis; conjunctivitis; contact dermatitis; Collin's ulcer; library Hing's ulcer; cystitis; gangrene; gingivitis; mastitis; esophagitis; pancreatitis; panniculitis; cellulitis gastritis; glomerulonephritis; and autoimmune diseases, including but not limited to inflammatory bowel Ulcerative colitis, Crohn's disease, Addison's disease, and glomerulonephritis (eg, crescentic glomerulonephritis, proliferative glomerulonephritis).

Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

Description of drawings

The following "detailed description" given by way of example is not intended to limit the present invention to the specific embodiments described, which can be understood in conjunction with the drawings incorporated by reference herein.

Figure 1 shows the diffuse expression of Lgr5-GFP in vivo. Small intestines were harvested from Lgr5-GFP mice and imaged directly under a fluorescence microscope. Although all areas of the small intestine were covered by crypts, approximately half of these crypts contained GFP+ cells. Scale bar: 100 μm.

Figures 2A-2H show combinations of CHIR and VPA that promote proliferation and self-renewal of Lgr5+ stem cells. Figure 2A shows the culture in the presence of ENR (EGF, noggin and R-spondin1), ENR+VPA (ENR-V), ENR+CHIR (ENR-C) and ENR+VPA+CHIR (ENR-CV) GFP and bright-field images of intestinal crypts at 6 days. Apoptotic cells are visible in the lumen with autofluorescence (red arrows), while white arrows indicate specific Lgr5-GFP at the bottom of the crypts. Scale bar: 100 μm. Figure 2B demonstrates the quantification of cell proliferation and GFP expression of crypts cultured under various conditions. Crypts were cultured in 24-well plates for 6 days and dissociated into single cells using Accutase. Viable cells in each well were counted as an indicator of cell proliferation. Lgr5-GFP expression was determined by flow cytometric analysis. Error bars represent standard deviation (S.D.) of triplicate wells. The experiment was performed 3 times and showed similar results. Figures 2C and 2D show flow cytometric analysis of GFP expression of individual Lgr5-GFP cells cultured for 7 days under the various conditions indicated. Error bars represent standard deviation of triplicate wells. Figure 2E shows GFP and bright field images of a single Lgr5-GFP cell after 9 days of culture. Scale bar: 100 μm. Figure 2F shows representative images of 4,000 FACS isolated single Lgr5-GFP cells after 7 days of culture under various conditions, while Figure 2G shows quantification of colony numbers. Figure 2H demonstrates metaphase expansion of cells cultured under CV conditions for 80 days with normal karyotype (2n=40). (Unless otherwise indicated, in all figures: ***P<0.001; **P<0.01; *P<0.05; NS P>0.05)

Figures 3A-3G demonstrate cell growth and GFP expression as a function of culture conditions. Figures 3A and 3B show the number of colonies and viable single cells from triplicate wells enumerated at each time point, respectively. Error bars indicate standard deviation. In Figure 3A, the series from left to right are Day 0, Day 2, Day 4, Day 6, Day 8 and Day 10. Figure 3C demonstrates FACS sorting of freshly isolated single Lgr5-GFP+ cells. Collect GFP ^-high single cell populations. Representative FACS analysis and gating strategy to define the GFP+ cell population are shown. Freshly isolated single cells from crypts show two differential GFP ^high and GFP ^low populations, whereas cultured cells do not show differential GFP ^high and GFP ^low populations, thereby gating on all GFP+ cells (gate) for analysis. Note that ENR-CV cultured cells show a single GFP+ population that is highly positive for GFP. The GFP- population represents Lgr5- cells as well as Lgr5+/GFP- cells (i.e., GFP-silenced stem cells), which are present in all unsorted crypt tissues but not in sorted single Lgr5-GFP cell cultures in (see Figure 2C). A total of 10,000 live cells were analyzed for each sample. Figure 3D demonstrates growth factor requirements for self-renewal of Lgr5+ stem cells under CV culture conditions. Crypts were cultured with EGF, noggin, R-spondin 1 and their combinations in the presence of CHIR and VPA for 6 days, as indicated. E: EGF (50ng/ml); N: noggin (100 ng/ml); R: R-spondin 1 (500ng/ml); C: CHIR (3μΜ); V: VPA (1mM). Figure 3E shows crypts cultured for 6 days under the various conditions indicated. GFP and bright field images are shown. Scale bar: 200 μm. Figure 3F shows the morphology and Lgr5-GFP expression of colonic crypts cultured under ENR, ENR-C and ENR-CV conditions. Figure 3G demonstrates the isolated number of organoids formed at day 5 in the presence of various concentrations of EGF, noggin, and R-spondin1 or R-spondin 2. All scale bars: 200 μm.

Figures 4A-4B demonstrate testing of various culture conditions for EPHB2+ human colonic stem cells. Crypts were cultured for 6 days under various conditions as indicated. GFP and bright field images are shown in Figure 4A. W: Wnt3a (100 ng/ml); Ni: Nicotinamide (10 mM); P: PGE2 (0.02 μΜ); A: A-83-01 (0.5 μΜ); S: SB202190 (10 μΜ); acid VPA (1 mM). EGF, Noggin, R-spondin 1, Wnt3a and VPA or ENR-W-V, conditions served as controls to show an increase in GFP expression. Scale bar: 200 μm. Figure 4B demonstrates the quantification of cell proliferation and GFP expression of crypts cultured under various conditions. Crypts were cultured in 24-well plates for 6 days and dissociated into single cells. The number of viable cells in each well was counted and the percentage of GFP+ cells analyzed by flow cytometry. Error bars represent standard deviation of triplicate wells.

Figures 5A-5D show cultures of single Lgr5-GFP stem cells. Figure 5A shows a single isolated Lgr5-GFP+ cell cultured for 9 days under CV conditions. Scale bar: 200 μm. Figure 5B shows 1500 FACS sorted single Lgr5+ cells cultured in Matrigel under the indicated conditions. Representative images from day 7 cultures are shown. Figure 5C demonstrates quantification of colony numbers. Error bars indicate standard deviation of triplicate wells. Figure 5D shows sorted single Lgr5+ stem cells seeded in a 48-well plate. Viable cell numbers were quantified 12 hours after plating. The number of colonies was counted on day 7 and the efficiency of colony formation was quantified. V: VPA; C: CHIR; W: Wnt3a at 100 ng/ml. Error bars represent standard deviation of triplicate wells. Experiments were performed 3 times and showed similar results.

Figures 6A-6D demonstrate maintenance of Lgr5+ stem cell self-renewal by the combination of CHIR and VPA. Shown are organoids cultured under ENR conditions (upper panels) and colonies cultured under ENR-CV conditions (lower panels) for Lysozyme (Figure 6A), Ki67 (Figure 6B) and EdU (Figure 6C) staining. Confocal image. For EdU staining, cells were incubated with the thymidine analog Edu (red) for 1 hour. In Figures 6B and 6C, only the crypt region contained Ki67-positive cells or incorporated EdU under ENR conditions (upper panels), whereas under CV conditions Ki67 or EdU was present throughout cell aggregates (lower panels). Figure 6D shows quantitative real-time PCR analysis of relative mRNA expression of markers for mature intestinal epithelial cells cultured for 6 days under the indicated conditions (intestinal alkaline phosphatase [Alpi] for intestinal epithelial cells, for goblet cells are mucin 2 [Muc2], chromogranin A [ChgA] for enteroendocrine cells, lysosomes [Lyz] for Paneth cells, and Lgr5 for small intestinal stem cells). ENR-CV(D40) indicates cells cultured under CV conditions for 40 days. Scale bar: 50 μm. In Figure 6D, the series from left to right are Alpi, Muc2, ChgA, Lyz and Lgr5.

Figures 7A-7D demonstrate the differentiation of intestinal stem cells cultured under CV conditions. Figure 7A shows the staining of differentiation markers (Alp for intestinal epithelial cells, Muc2 for goblet cells (white arrows) and mucin secreted by goblet cells, for cells transferred from CV conditions to ENR conditions and cultured for 4 days, ChgA for enteroendocrine cells and Lyz for Paneth cells). Nuclei were stained with DAPI and GFP indicated the presence of stem cells. Figure 7B shows real-time RT-PCR analysis of relative mRNA expression of mature intestinal epithelial markers from cells cultured under various conditions. Cell colonies were then harvested, washed and replated into wells of 24-well plates and cultured in Matrigel for 4 days under various conditions as indicated. ENR was added under all conditions, and cells cultured with ENR alone were used as controls. I: IWP-2 (2 μΜ), D: DAPT (10 μΜ), C: CHIR (3 μΜ), V: VPA (1 mM). Error bars represent standard deviation. Figure 7C demonstrates Alp staining of cells cultured under various conditions. Cells under ID and CD conditions showed marked morphological changes, similar to goblet and Paneth cells. Scale bar: 50 μm. Figure 7D demonstrates immunocytochemical staining for markers of differentiation. Cells cultured under CD and ID conditions were used for mucin 2 (Muc2), chromogranin A (ChgA) and lysosome (Lyz) staining. Three-dimensionally reconstructed confocal images are shown. Scale bar: 50 μm.

Figures 8A-8F demonstrate the controlled differentiation of Lgr5+ stem cells in vitro. Figure 8A demonstrates staining of organoids cultured under ENR conditions. Left panels demonstrate Alp staining of intestinal epithelial cells. Before staining, the organoids were dissected with a sharp blade and the luminal contents removed under a dissecting microscope. The middle panel shows Muc2 staining of goblet cells (arrows) and mucin secreted by goblet cells, while the right panel shows ChgA staining of enteroendocrine cells. GFP+ cells indicate Lgr5+ stem cells. Figure 8B provides a schematic representation of the differentiation strategy. Single Lgr5+ stem cells were cultured under CV conditions for 4 to 6 days to form colonies. Cell colonies were then harvested, washed and embedded into fresh matrigel, and cultured under various conditions. Figure 8C shows the morphology of cell colonies transferred from CV to ENR conditions and cultured for 4 days (upper panel). Colonies cultured continuously under CV conditions are shown as controls (lower panel). Figure 8D shows the morphology of differentiated cells for each condition at low to high magnification images. Note the distinct changes in the morphology of most cells under CD and ID conditions, reflecting the formation of Paneth cells and goblet cells, respectively. Figure 8E demonstrates Alp staining of colonies cultured under IV conditions. Apical staining (left panel) and uniform staining (right panel) of Alp are shown. Figure 8F demonstrates Muc2 staining of colonies cultured under ID and CD conditions. All scale bars: 50 μm.

Figures 9A-9F illustrate the mechanism of action of CHIR and VPA. Figure 9A shows the morphology and Lgr5-GFP expression of crypts cultured for 6 days under various conditions. C: CHIR (3 μΜ); Li: LiCl (5 mM); W: Wnt3a (100 nM). Figure 9B shows the cell number and percentage of GFP+ cells of 6-day crypt cultures. Data represent three independent experiments. Figure 9C shows 6-day crypt cultures under ENR-C (control) conditions or with HDAC inhibitors. Figure 9D shows the quantification of GFP percentage, total viable cell number and relative GFP intensity for the cells in Figure 9C. Figure 9E shows the effect of VPA and TSA on cell proliferation and GFP expression at various concentrations. Figure 9F demonstrates the effect of nicotinamide (Ni) in combination with Wnt3a (W, 100 ng/ml) or CHIR (C, 3 μΜ). Cell numbers and percentage of GFP+ cells are shown for crypts cultured for 6 days under various conditions (unless otherwise noted, in all figures: Error bars represent standard deviation or triplicate wells. ***P<0.001 ; **P<0.01; *P<0.05; NS P>0.05).

Figure 10 shows the morphology and GFP expression of individual Lgr5-GFP cells cultured under various conditions. Scale bar: 100 μm.

Figures 11A-11D illustrate the mechanism of VPA. Figure 11A demonstrates that VPA rescues GFP expression after Notch inhibition. Crypts were cultured for 3 days under ENR-C conditions in the presence or absence of DAPT (D, 5 μΜ) and different concentrations of VPA (V, 0.25 mM-4 mM). Scale bar: 200 μm. Figures 11B and 11C show crypts cultured for 4 days under ENR (Fig. 11B) or ENR+CHIR (Fig. 11C) conditions followed by addition of different concentrations of VPA for an additional 24 hours. Expression of Notch1, Hes1 and Atoh1 was analyzed by real-time RT-PCR. Figure 11D shows the analysis of the expression of Notch1, Hes1 and Atoh1 by real-time RT-PCR in crypts after 6 days of culture. In Figures 11B-11C, the series from left to right is 0, 0.5, 1, 2 and 3. In Fig. 1 ID, the series from left to right are ENR, ENR-V, ENR-C and ENR-CV.

Figures 12A-12B demonstrate a model of intestinal stem cell self-renewal and differentiation under physiological conditions (Figure 12A) and in in vitro culture (Figure 12B).

Figures 13A-13B demonstrate the combination of CHIR and VPA that promote proliferation and GFP expression of Lgr5+ stem/progenitor cells derived from the mouse inner ear. Figure 13A shows bright field and GFP images of isolated cochlear sensory epithelium derived from postnatal day 2 Lgr5-GFP mice. Figure 13B shows isolated cochlear sensory epithelium dissociated into single cells and cultured for 11 days under various conditions. E: EGF; N: noggin; R: R-spondin 1; C: CHIR99021, V: VPA. Scale bar: 100 μm.

Figures 14A-14F demonstrate the combination of CHIR and VPA that promote proliferation and GFP expression of Lgr5+ stem/progenitor cells derived from the mouse inner ear. Figure 14A demonstrates GFP expression in inner ear epithelial cells. Figure 14B shows quantification of GFP expression and cell number. Figure 14C shows bright field and GFP images. Figure 14D shows the cell numbers of 8-day cultures of inner ear stem cells under the various conditions indicated. Figure 14E shows the percentage of GFP for 8-day cultures of inner ear stem cells under the various conditions indicated. Figure 14F demonstrates the morphology and GFP expression of Lgr5-GFP inner ear stem cells cultured under various conditions. All scale bars: 200 μm.

Figure 15 shows the in vitro inoculation of murine intestinal crypts in healthy mouse colon tissue. The left image shows isolated small intestinal crypts placed on the colon with partially exposed epithelium. White arrows indicate the inoculated crypts. The right panel shows the inoculated crypts attached to the colon and spread on its surface for 24 hours. The black arrows point to the same locations as the white arrows in the illustration on the left.

Figure 16 demonstrates crypt engraftment 48 hours after inoculation. Fluorescence (upper panel) and bright-field (lower panel) images of mouse colon tissue inoculated with crypts are shown. Crypts were stained with DiD before inoculation. White lines indicate areas including implanted cells.

Figure 17 shows crypt engraftment after 6 days of in vitro culture. GFP (left panel), RFP (middle panel) and bright field (right panel) channel images of mouse colon tissue inoculated with crypts are shown. GFP signal indicates the presence of Lgr5 cells.

Figure 18 shows confocal images of prolapsed ulcerative colitis tissue excised from TRUC mice with implanted crypts. Crypts were stained with DiD before inoculation. Prolapsed tissue is shown in green autofluorescence.

Figures 19A-19N show schematic representations of inoculation (left) and post-incubation organoid growth (right) in the culture systems evaluated. Figures 19A and 19B demonstrate a typical submucosal seeding method (referred to herein as "bare patch") that supports monolayer growth and organoid dissociation. Figures 19C and 19D demonstrate GF-infused SIS supporting 3D organoid growth (GF includes EGF, noggin, R-spondin 1, Y-27632, valproic acid, CHIR). Figures 19E and 19F show gel patches consisting of GF-infused SIS overlaid with collagen. Figure 19E inset shows individual organoids encapsulated individually in soft gels and SIS substrates. Figures 19G and 19H show typical collagen suspensions with GFs (EGF, noggin, R-spondin 1, Y-27632, valproic acid, CHIR) added directly to the medium. Figures 19I and 19J show typical collagen suspensions with GF (EGF, noggin, R-spondin 1, Y-27632, valproic acid, CHIR) embedded in the gel. Figures 19K and 19L show typical collagen suspensions without additional GF added to the medium. Figures 19M and 19N show a typical Matrigel suspension without additional GF added to the medium (experimental control).

Figure 20A shows a schematic of the seeding procedure with Lgr5+ organoids; patterned circles represent infused growth factors (EGF, noggin, R-spondin 1, Y-27632, valproic acid, CHIR). Figure 20B shows the initial adherent phase, with arrows indicating a diffusion support for entrapped growth factors. Figure 20C shows a complete culture system with collagen overlays demonstrating thickness measurements.

Figure 21A provides a comparison of organoid growth in seven culture systems. The series from left to right are Matrigel, Gel-Patch with GF, Bare Patch with GF, Collagen I, Collagen I with Media GF, Collagen with Embedded GF Protein I and bare patch without GF. Figures 21B and 21C show representative organoids at 48 hours from the gel patch system with GF and GFP+ fluorescence indicating the presence of Lgr5+ stem cells at the bottom of the crypts (partial central autofluorescence visible). 24 hours (collagen I (CI) vs. all), 48 hours (bare patch with GF (BPGF) vs. all; CI vs. Matrigel (M); CI with GF (CIGF) vs. M, CI, collagen I with embedded GF (CIEGF), bare patch (BP), gel patch system with GF (PSGF)), 72 hours (CI vs. all; BP, CIEGF and CIGF vs. M, PS, BPGF, CI), at 96 hours (CI vs. all, BP, CIEGF and CIGF vs. all) *=p<0.05. Scale bar (FIGS. 21B and 21C) = 200 [mu]m.

Figure 22 demonstrates successful growth and crypt expansion of seeded organoids in the gel patch system with GF. A series of representative graphs demonstrating ex vivo expansion of seeded organoids on the SIS patch system is shown. Out-of-focus crypts are the effect of three-dimensional growth observed under a single plane microscope.

Figure 23A provides a schematic showing the establishment of a 4 mm gastric defect. A 6mm patch is shown placed over the defect in Figure 23B. As shown in Figure 23C, there were no visible defects on the outer stomach wall as indicated on a representative stomach sample 1 week post-surgery. Severe defects viewed from the inner gastric wall (arrows) are demonstrated according to the type of patch placed: Figure 23D shows the SIS patch without GF, Figure 23E shows the SIS patch+GF, and Figure 23F shows the SIS without PGSU backing only. The SIS patch with GF showed complete closure and epithelialization of the gastric wall defect, whereas the defect remained partially open in SIS alone and completely open in PGSU without SIS.

Figure 24 shows real-time RT-PCR analysis of marker gene expression in isolated human intestinal crypts cultured under various conditions. EGF, noggin and R-spondin 1 were added in all conditions. C: CHIR, Ni: Nicotinamide, W: Wnt3a, A: A83-01, S: SB202190, P: PGE2, V: VPA, Tu: Tubastatin A, crypts indicate freshly isolated human small intestinal crypts. Error bars indicate standard deviation, n=3.

Figures 25A-25B demonstrate optimized culture conditions for human intestinal stem cells. Figure 25A demonstrates the proliferation of human intestinal epithelial cells cultured under various conditions. Freshly isolated human intestinal crypts were cultured under various conditions as indicated. EGF, noggin and R-spondin 1 were present in all conditions. Cell numbers were quantified on day 9 post seeding. C: CHIR, V: VPA used at 0.5-1.5 mM, Ni: Nicotinamide. Figure 25B shows LGR5 expression in cells cultured under various conditions as in Figure 25A. Use 1 mM VPA.

Figure 26 shows human intestinal stem cell cultures. Cells were cultured in human intestinal stem cell medium (containing EGF, noggin, R-Spondin1, CHIR99021, VPA and nicotinamide). Passage 2 cells on day 5 after passaging are shown. Scale bar: 400 μm.

Figure 27 demonstrates the increase in crypt size following in vivo administration of CHIR and VPA over the course of 7 days in an animal model system.

Detailed ways

definition

As used herein, an "antibody" is any immunoglobulin polypeptide or fragment thereof that has the ability to bind an immunogen.

As used herein, an "agonist" is an agent that results in increased expression or activity, respectively, of a target gene or protein. An agonist can bind and activate its cognate receptor in some form, which directly or indirectly brings about a physiological effect on the target gene or protein.

As used herein, an "inhibitor" is an agent that results in a decrease in the expression or activity, respectively, of a target gene or protein. An "antagonist" may be an inhibitor, but is more specifically an agent that binds to a receptor, which in turn reduces or eliminates binding to other molecules.

As used herein, an "inhibitory nucleic acid" is a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in decreased expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule or an ortholog thereof, or comprises at least a portion of a complementary strand of a target nucleic acid molecule. Typically, expression of the target gene is reduced by 10%, 25%, 50%, 75%, or 90%-100%.

"Antisense" refers to a nucleic acid sequence that is complementary to the coding strand of a nucleic acid sequence or mRNA, regardless of its length. As referred to herein, a "complementary nucleic acid sequence" is a nucleic acid sequence capable of hybridizing to another nucleic acid sequence consisting of complementary nucleotide base pairs. "Hybridization" refers to the pairing between complementary nucleotide bases to form double-stranded molecules under suitable stringent conditions (for example, in DNA, adenine (A) forms a base pair with thymine (T), and guanine (G) forms a base pair with cytosine (C)) (see, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507) . In one embodiment, antisense RNA is introduced into individual cells, tissues or organoids. Antisense nucleic acids may contain modified backbones, such as phosphorothioate backbones, phosphorodithioate backbones, or other modified backbones known in the art, or may contain non-natural internucleoside linkages.

"siRNA" refers to double-stranded RNA. Optimally, the siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3' end. These dsRNAs can be introduced into individual cells or culture systems. Such siRNAs are used to downregulate mRNA levels or promoter activity.

As used herein, a "fragment" is a portion of a polypeptide or nucleic acid molecule. The portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides or amino acids.

As used herein, the term "stem cell" refers to a pluripotent cell that has the ability to self-renew and differentiate into a variety of cell lineages.

As used herein, the term "epithelial stem cell" refers to a pluripotent cell that has the potential to commit to a variety of cell lineages, including cell lineages that give rise to epithelial cells.

As used herein, the term "progenitor cell" refers to a lineage-restricted cell derived from a stem cell.

As used herein, the term "epithelial progenitor cell" refers to a pluripotent cell that has the potential to become a lineage of cells restricted to the generation of epithelial cells.

As used herein, the term "self-renewal" refers to the process by which a cell divides to produce one (asymmetrical division) or two (symmetrical division) daughter cells with developmental potential, the developmental potential of which is the same as There was no difference in the developmental potential of the mother cells. Self-renewal involves proliferation and maintenance of an undifferentiated state.

As used herein, the term "implantation" refers to the process of in vivo incorporation of stem or progenitor cells into a tissue of interest by contact with the existing cells of the tissue.

As used herein, the term "isolated" means free to varying degrees of components normally associated with it found in its natural state. "Isolated" refers to the degree of separation from the original source or environment.

As used herein, the term "population" of cells is any number of cells greater than 1, but preferably at least 1 x ¹⁰³ cells, at least ¹ x 104 cells, at least ¹ x 105 cells, at least 1 x 106 cells, at least ¹ x ¹⁰⁷ cells, at least ¹ x 108 cells, at least 1 x ¹⁰⁹ cells, or at least ¹ x 1010 cells.

As used herein, the term "organoid" or "epithelial organoid" refers to a cluster or aggregate of cells that resembles an organ or a part of an organ and possesses cell types associated with that particular organ.

As used herein, a "subject" is a vertebrate including any member of the class Mammalia.

As used herein, a "mammal" is any mammal including, but not limited to, a human, mouse, rat, sheep, monkey, goat, rabbit, hamster, horse, cow, or pig.

As used herein, "non-human mammal" refers to any mammal that is not a human.

As used herein, "increase" means, for example, an increase of at least 5%, such as 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, compared to a reference level , 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% or more.

As used herein, "increase" also refers to an increase of at least 1-fold, such as 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, e.g. 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 500 times, 1000 times or more.

As used herein, "reduce" means, for example, a reduction of at least 5%, such as 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, compared to a reference level , 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%.

As used herein, "reduction" also refers to at least 1-fold reduction, such as 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, e.g. compared to a reference level , 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 500 times, 1000 times or more.

As used herein, the term "reference" refers to a standard or control condition (eg, not treated with a test reagent or combination of test reagents).

As used herein, the term "eliminate" means to reduce to an undetectable level.

As used herein, the term "synergy" or "synergistic effect" refers to an effect that is greater than the sum of each effect when taken individually; it is greater than the additive effect.

As used herein, the term "treating" and the like refers to reducing or alleviating a condition and/or symptoms associated therewith. It is to be understood that treatment of a disorder or condition does not require, but does not preclude, the complete elimination of that disorder, condition or symptoms associated therewith.

In the present disclosure, "comprising", "containing" and "having" etc. may have the meanings assigned to them in the US Patent Law, and may refer to "comprising" etc.; the meaning given to it, and the term is open-ended such that there may be more than stated, provided that the essential or novel characteristics of said stated are not altered by the presence of more than stated, but excluding Prior art implementation.

Other definitions appear in the context of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

Methods and compositions of the invention

I. Cell Culture Solutions and Systems

Cell culture solutions and systems have now been discovered that promote homogeneous epithelial stem cell culture, efficient epithelial organoid formation, and their scale-up for transplantation.

Inhibitors including bone morphogenetic proteins, inhibitors of glycogen synthase kinase-3β (GSK3β), agents that bind leucine-rich repeat G protein-coupled receptor 5 (LGR5), and histone deacetylation can be utilized A cell culture solution containing enzyme inhibitors was used to form epithelial cell colonies from isolated epithelial stem cells. In specific embodiments, at least about 25%, at least about 40%, at least about 50%, at least about 75%, at least about 90% to about 100% of the isolated epithelial stem cells form an epithelium in the presence of the cell culture solution Cell colonies. Additionally, at least about 6% of the individually isolated epithelial stem cells form epithelial cell colonies in the presence of the cell culture solution. 1,6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl) as an inhibitor of glycogen synthase kinase-3β The combination of )-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile "CHIR99021" (Ring et al., 2003) and valproic acid as a histone deacetylase inhibitor had a positive effect on colony formation efficiency. Synergy.

Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily and include metalloproteinases involved in embryonic patterning across species and in postembryonic cell signaling. Inhibitors of BMPs include, for example, agents that bind to a BMP molecule to form a complex in which the activity of the BMP is reduced or eliminated (eg, by preventing or inhibiting the binding of the BMP molecule to a BMP receptor). Alternatively, the inhibitor is an agent that acts as an antagonist or inverse agonist. Such inhibitors bind to the BMP receptor and prevent the binding of BMP to the receptor. An example of the latter agent is an antibody that binds a BMP receptor and that prevents BMP from binding to the receptor to which the antibody binds. Inhibitors of BMP are well known in the art (Rider et al., 2010) and may include, but are not limited to, noggin, chordin, follistatin (Schneyer et al., 1994), DAN, including DAN Cysteine knot domain proteins (including Cerberus and Gremlin), sclerostin, gastrulation protein, uterine sensitivity-related gene-1, connective tissue growth factor (Abreu et al., 2002), inhibin (Wiater and Vale , 2003), BMP-3 (Gamer et al., 2005), Dorsomorphin (Yu et al., 2008) and derivatives including DMH1 (Hao et al., 2010) and LDN-193189 (Cuny et al., 2008).

Glycogen synthase kinase-3 (GSK3) is a proline-directed serine-threonine kinase that was originally identified as phosphorylated and Inactive glycogen synthase. Wnt agonists comprising GSK-3β inhibitors are well known in the art and include, but are not limited to: 1,6-[[2-[[4-(2,4-dichlorophenyl)-5-(5 -Methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile "CHIR99021" (Ring et al., 2003), LiCl (Klein et al., 1996), BIO -acetone oxime ((2'Z,3'E)-6-bromoisatin-3'-oxime) (Meijer et al., 2003), N6-[2-[[4-(2,4-dichlorophenyl )-5-(1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine "CHIR98014" (Ring et al., 2003), 3-( 2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione "SB 216763" (also known as GSK-3 inhibitor IV) (Coghlan et al., 2000), 3-[(3-Chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione "SB 415286 "(Coghlan et al., 2000), 5-ethyl-7,8-dimethoxy-1H-pyrrolo[3,4-c]-isoquinoline-1,3-(2H)-dione"3F8 "(Zhong et al., 2009), 9-bromo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one "Campalone" ( Schultz et al., 1999; Zaharevitz et al., 1999), 9-bromo-7,12-dihydro-pyrido[3',2':2,3]azepine[4,5-b]indole-6( 5H)-keto "1-azacaprolone" (Schultz et al., 1999; Zaharevitz et al., 1999), N-(3-chloro-4-methylphenyl)-5-(4-nitrophenyl )-1,3,4-oxadiazol-2-amine "TC-G 24" (Khanfar et al., 2010), 2-methyl-5-[3-[4-(methylsulfinyl)phenyl ]-5-benzofuryl]-1,3,4-oxadiazole "TCS 2002" (Saitoh et al., 2009), N-[(4-methoxyphenyl)methyl]-N'-( 5-nitro-2-thiazolyl)urea "AR-A 014418" (Bhat et al., 2003), 3-[5-[4-(2-hydroxy-2-methyl-1-oxypropyl)- 1-piperazinyl]-2-(trifluoromethyl)phenyl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione "TCS 21311" (Thoma et al. 2011), 3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]-phenol "TWS 11 9" (Ding et al., 2003), ((2'Z,3'E)-6-bromoisatin-3'-acetone oxime) "BIO-acetone oxime" (also known as GSK-3 inhibitor IX) ( Meijer et al., 2003), 4-(2-amino-4-oxyl-2-imidazolin-5-ylidene (ylidene))-2-bromo-4,5,6,7-tetrahydropyrrolo[ 2,3-c] azepin-8-one "10Z-Hymenialdisine" (Breton et al., 1997), 2-[(3-iodophenyl)methylsulfanyl]-5-pyridine -4-yl-1,3,4-oxadiazole (also known as GSK-3 inhibitor II) (Wada, 2009), 4-benzyl-2-methyl-1,2,4-thiadiazole Alkane-3,5-dione (also known as GSK-3 inhibitor I) (Wada, 2009), 3-amino-6-(4-((4-methylpiperazin-1-yl)sulfonyl) Phenyl)-N-(pyridin-3-yl)pyrazine-2-carboxamide (HCl salt) (also known as GSK-3β inhibitor XXVII) (US Patent Publication No. 2006/0173014), 4,5-di (1-Methyl-1H-indol-3-yl)-1,2-dihydropyrazol-3-one (also known as GSK-3β inhibitor XXVI) (Chen et al., 2011), FRATtide peptide SQPETRTGDDDPHRLLQQLVLSGNLIKEAVRRLHSRRLQ( SEQ ID NO: 1) (Bax et al., 2001), 3-amino-1H-pyrazolo[3,4-b]quinoxaline "Cdk1/5 inhibitor" (Andreani et al., 1996, 2000; Katoh et al., 2011 ) and 4-((5-bromo-2-pyridyl)amino)-4-oxobutanoic acid "Bikinin" (DeRybel et al., 2009). Preferably, the GSK-3 inhibitor is CHIR99021.

Leucine-rich repeat G protein-coupled receptor 5 (LGR5) is known for its restricted crypt expression and marker of stem cells in a variety of adult tissues and cancers. Agents that bind to the LGR5 receptor include, but are not limited to, R-spondins (Kim et al., 2006; Nam et al., 2006), such as R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4. Preferably, the agent that binds to the LGR5 receptor is R-spondin 1.

In alternative embodiments, lithium chloride (LiCl) can be substituted for CHIR99021, or R-spondin 1 can be substituted with at least about 3 μm of CHIR99021.

Histones are nuclear proteins that bind DNA and form nucleosomes. It is directly involved in the packaging of DNA into chromosomes and the regulation of transcription. Histone acetylation/deacetylation is a major factor regulating the dynamics of chromatin structure during transcription. Histone deacetylase inhibitors that reduce or eliminate histone deacetylation are well known in the art and can include, but are not limited to, Pan-HDAC inhibitors (e.g., valproic acid, trichostatin A, suberoyl Suberoylanilide hydroxamic acid and suberoylhydroxamic acid (SBHA) and HDAC6 inhibitors (eg, Tubacin, Tubastatin A, and compound 7) .

In alternative embodiments, Atoh1 inhibitors may augment or replace histone deacetylase inhibitors. Atoh1 inhibitors include, for example, inhibitory nucleic acids that result in the reduction or elimination of Atoh1 expression. Inhibitory nucleic acids targeting Atoh1 are known in the art (Shi et al., 2010).

The cell culture solution may optionally include epidermal growth factor and/or a Notch agonist. Epidermal growth factor is a cell signaling molecule involved in a variety of cellular functions including cell proliferation, differentiation, motility, and survival, as well as tissue development. Notch proteins are one-way transmembrane receptors that regulate cell fate decisions during development. Notch agonists include, for example, agents that increase Notch activity in a cell. Notch agonists are well known in the art and may include, but are not limited to, Notch 1 antibody (N1 Ab), Delta 1, Delta-like 3, Delta-like 4, Jagged 1, Jagged 2, DSL peptide, and Delta D.

In a specific embodiment, the cell culture solution comprises about 5 ng/ml to about 500 ng/ml EGF, about 5 ng/ml to about 500 ng/ml of noggin, about 50 ng/ml to about 1000 ng/ml of R-spondin, about 0.1 μM to about 10 μM CHIR99021 and about 0.1 mM to about 5 mM valproic acid.

In other embodiments, a combination of a Wnt agonist and an HDAC6 inhibitor in a cell culture solution is preferred. Accordingly, the cell culture solution may include an inhibitor of bone morphogenetic protein, R-spondin 1, a Wnt agonist, and an HDAC6 inhibitor.

Wnt proteins are extracellular signaling molecules involved in the control of embryonic development. Wnt agonists are well known in the art and include, but are not limited to: Wnt-1/Int-1 (Nusse et al., 1982), Wnt-2/Irp (Int-1-related protein) (Wainwright et al., 1988), Wnt -2b/13 (Katoh et al., 1996), Wnt-3/Int-4 (Katoh et al., 2001), Wnt-3a (Saitoh et al., 2001), Wnt-4 (Smolich et al., 1993), Wnt-5a (Burrus et al. et al., 1995), Wnt-5b (Burrus et al., 1995), Wnt-6 (Burrus et al., 1995), Wnt-7a (Smolich et al., 1993), Wnt-7b (Burrus et al., 1995), Wnt-8a/8d (Saitoh et al., 2001), Wnt-8b (Lako et al., 1998), Wnt-9a/14 (Bergstein et al., 1997), Wnt-9b/14b/15 (Bergstein et al., 1997), Wnt-10a (Wang et al., 1996), Wnt-10b/12 (Wang et al., 1996), Wnt-11 (Lako et al., 1998), Wnt-16 (Bergstein et al., 1997; Fear et al., 2000), R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, Norrie disease protein (Planutis et al., 2007), CHIR99021, LiCl, BIO ((2'Z,3'E)-6-bromoisatin-3'-oxime), CHIR98014 , SB 216763, SB 415286, 3F8, Kemparolone, 1-Azakenparolone, TC-G24, TCS 2002, AR-A 014418, 2-Amino 4-[3,4-(Methylenebis Oxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine (Liu et al., 2005), 2-[2-(4-acetylphenyl)diazenyl]-2-(3 ,4-dihydro-3,3-dimethyl-1(2H)-isoquinolinylidene)acetamide "IQ 1" (Miyabayashi et al., 2007), (3α,5β,12α,20R)-3, 12-Dihydrochol-24-alkanoic acid "DCA" (Pai et al., 2004), (2S)-2-[2-(indan-5-yloxy)-9-(1,1'-biphenyl -4-yl)methyl]-9H-purin-6-ylamino]-3-phenyl-1-propanol "QS 11" (Zhang et al., 2007), piperidinyl diphenylsulfonylsulfonamide 1 "WAY-316606" (Bodine et al., 2009), (hetero)arylpyrimidines (Gilbert et al., 20 10), 10Z-Hymendexide, TCS 21311, TWS 119, GSK-3β Inhibitor II, GSK-3β Inhibitor I, GSK-3β Inhibitor XXVII, GSK-3β Inhibitor XXVI, FRATtide, Cdk1/5 Inhibitor and Bikinin.

A cell culture system includes a cell culture solution of the invention and epithelial organoids, epithelial stem or epithelial progenitor cells, or a population of epithelial stem or epithelial progenitor cells. Epithelial organoids are known in the art (Yao et al., 2010; Lukacs et al., 2010). Epithelial stem cells include, but are not limited to, stem cells of the small intestine, stomach, lung, pancreas, and colon. Epithelial stem cells also include LGR5 positive stem cells derived from including but not limited to small intestine, inner ear, brain, kidney, liver, retina, stomach, pancreas, breast, hair follicle, ovary, adrenal medulla, skin, thymus, taste buds, mammary gland, Source of malignancy and tumors. Epithelial stem cells also include quiescent precursors of LGR5-positive stem cells that express LGR5 (Buczacki et al., 2013). The population of epithelial stem cells or epithelial progenitor cells in the cell culture system may comprise, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the %, 99% or 100% cells. Preferably, the population of epithelial stem cells or epithelial progenitor cells is maintained during repeated passages.

In specific embodiments, human epithelial stem cells can be cultured in the presence of additional components including nicotinamide or a Sirt1 specific HDAC inhibitor (eg EX527).

In specific embodiments, epithelial stem cells derived from the inner ear can be cultured in the presence of Wnt agonists, histone deacetylase inhibitors, epidermal growth factor, basic fibroblast growth factor, and optionally bone morphogenetic protein .

The cell culture system may include additional components including, but not limited to, a submucosa substrate and a collagen-containing coating to form a three-dimensional tissue construct suitable for transplantation. The collagen coating can overlie and/or surround selected epithelial tissue or cell types, as well as be placed between the selected epithelial tissue or cell types and the submucosal substrate. Selected epithelial tissues or cell types include, but are not limited to, epithelial stem cells, isolated tissues comprising epithelial stem cells, or epithelial organoids.

Small intestinal submucosa (SIS) is a common biocompatible and clinically used scaffold (de la Fuente et al., 2003; Ueno et al., 2007; Schultz et al., 2002; Kehoe et al., 2012). Submucosal scaffolds undergo rapid neovascularization, particle formation, biodegradation, and are generally well conserved across species in terms of protein composition. Improved submucosa-based scaffolds for three-dimensional tissue constructs were prepared by inoculating the submucosa with preselected epithelial cell types and promoting growth with a collagen-based overlay. Altering the composition of SIS with this overlay promotes cell adhesion and growth of SIS wounds, leading to three-dimensional expansion of submucosa-adhered cells into large epithelial organoids. Animal-derived tissue matrix scaffolds from warm-blooded vertebrates (e.g., gastric submucosa, bladder submucosa, digestive submucosa, respiratory submucosa, reproductive submucosa, and hepatic basement membrane) are interchangeable with SIS and thus fall within the within the scope of this disclosure.

The tissue constructs may be cultured in the presence of cell culture solutions known in the art or as described herein above. For example, the tissue construct can be cultured in the presence of a cell culture solution comprising an inhibitor of bone morphogenetic protein, R-spondin 1, CHIR99021, and a histone deacetylase inhibitor. In addition, the submucosa matrix may comprise similar combinations of small molecules and/or growth factors including, but not limited to, epidermal growth factor, bone morphogenetic protein, R-spondin 1, CHIR99021, Y- 27632 and histone deacetylase inhibitors.

In an alternative embodiment, a collagen-free epithelial cell culture system is provided wherein the submucosa substrate contains, for example, epidermal growth factor, bone morphogenetic protein, R-spondin 1, CHIR99021, Y-27632, and histone deacetylase Combinations of small molecules such as inhibitors and/or growth factors. Collagen-free tissue constructs can be cultured in the presence of cell culture solutions known in the art or described above.

II. Methods of Utilizing Cell Culture Solutions and Systems

The cell culture solutions and systems of the present invention can be used to efficiently form epithelial organoids from isolated epithelial stem cells. In a specific embodiment, the isolated epithelial stem cells are incubated in the presence of noggin, R-spondin 1, CHIR99021 and a histone deacetylase inhibitor (e.g., valproic acid) at least about 25%, 35% , 40%, 50%, 60%, 70%, 80%, 90% or 100% efficiency formed epithelial cell colonies. In another specific embodiment, incubation of a single isolated epithelial stem cell in the presence of noggin, R-spondin 1, CHIR99021 and a histone deacetylase inhibitor forms at least about 6% to about 100% efficiency epithelial cell colonies.

Epithelial stem cells maintained within the cell culture solutions and systems of the invention can then be directed to specific differentiation pathways, including those that lead to the formation of Paneth cells, enterocytes, goblet cells, and enteroendocrine cells.

Paneth cells have been shown to be an important component of the Lgr5 ⁺ stem cell niche within intestinal crypts that provide critical signals for hepatocyte maintenance (Sato et al., 2011b; Yilmaz et al., 2012). Paneth cells are generated by first incubating epithelial stem cells in the presence of a cell culture solution containing inhibitors of BMP, R-spondin 1, CHIR99021, and histone deacetylase inhibitors (e.g., valproic acid), followed by further Epithelial stem cells are incubated in the presence of at least one Wnt agonist and at least one Notch inhibitor (eg, DAPT). Similarly, subsequently further incubating the epithelial stem cells in the presence of at least one Wnt inhibitor and at least one histone deacetylase inhibitor to generate intestinal epithelial cells; and subsequently further incubating the at least one Wnt inhibitor and and at least Epithelial stem cells were incubated in the presence of a Notch inhibitor to generate goblet cells. Wnt inhibitors can be, but are not limited to: N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno [3,2-d]pyrimidin-2-yl)thio]acetamide ("IWP-2") (Chen, Dodge et al., 2009). Notch can be, but is not limited to: N-[N-(3,5-difluorophenylacetyl)-L-alanyl]-S-phenylglycine tert-butyl ester ("DAPT" or "LY-374973") (Dovey, John et al., 2001), N1-[(7S)-6,7-dihydro-6-oxo-5H-dibenzo[b,d]azepine-7-yl]-2,2- Dimethyl-N3-(2,2,3,3,3-pentafluoropropyl)-("RO4929097", malonamide) (He, Luistro et al., 2011), (S)-2-hydroxy-3 -Methyl-N-((S)-1-((S)-3-methyl-2-oxo-2,3,4,5-tetrahydro-1H-benzo[d]azepine-1 -ylamino)-1-oxypropan-2-yl)butanamide ("LY450139") (Lanz, Hosley et al., 2004), N-[(1S)-2-[[(7S)-6,7-di Hydrogen-5-methyl-6-oxo-5H-dibenzo[b,d]azepine-7-yl]amino]-1-methyl-2-oxyethyl]-2-hydroxy-3- Methyl-,(2S)- ("LY900009", butyramide) Selleckchem: Cat. No. S7168, N-[(1S)-2-[[(7S)-6,7-Dihydro-5-(2-hydroxy Ethyl)-6-oxo-5H-pyrido[3,2-a][3]benzo[d]azepine-7-yl]amino]-1-methyl-2-oxyethyl]- 4,4,4-Trifluoro-("LY3039478", butyramide) Selleckchem: Cat. No. S7169, N-[(1S)-2-[[(7S)-6,7-Dihydro-5-methyl- 6-Oxo-5H-dibenzo[b,d]azepine-7-yl]amino]-1-methyl-2-oxyethyl]-3,5-difluoro-α-hydroxyl-,( αS)-("LY411575", phenylacetamide) (Wehner, Cizelsky et al., 2014), 7-(S)-[N'(3,5-difluorophenylacetyl)-L-alanyl]amino- 5-Methyl-5,7-dihydro-6H-dibenzo[b,d]azepine-6-one ("YO-01027" (DBZ)) (Milano, McKay et al., 2004), (2R )-2-(N-(2-fluoro-4-(1,2,4-oxadiazol-3-yl)benzyl)-4-chlorobenzenesulfonylamino)-5,5,5-trifluoro Pentamide ("BMS-708163") (Saito, Fu et al., 2014), (2R,3S)-N-[(3S)-1-methyl-2-oxo-5-phenyl-2,3-di Hydrogen-1H-1,4-benzodiazepine-3-yl]-2,3-bis(3,3,3-trifluoropropyl)succinamide ("BMS-906024") (Huang, Greer et al., 2009), (S,S)-2-[2-(3, 5-difluorophenyl)-acetylamino]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepine Heterazo-3-yl)-propionamide ("compound E") (Milano, McKay et al., 2004), 2-[(1R)-1-[[(4-chlorophenyl)sulfonyl](2,5 -difluorophenyl)amino]ethyl-5-fluorophenylbutyric acid ("BMS-299897") (Anderson, Holtz et al., 2005), SAHM1 Calbiochem Cat. No.: 491002, (Selective Abeta42) Calbiochem Cat. No.: 565792 and N-(2-bromophenyl)-N'-(2-hydroxy-4-nitrophenyl)urea ("SB 225002") (Bakshi, Jin et al., 2009).

Subsequent inactivation of at least one Notch inhibitor and inhibition of receptor tyrosine kinase (RTK), mitogen-activated protein (MAP) kinase (also known as MAPK/ERK) or extracellular signal-regulated kinase (ERK) (also known as Further incubation of the epithelial stem cells in the presence of at least one of MAPK/ERK) produces epithelial endocrine cells. MAP kinase may be, but not limited to, mitogen-activated protein (MAP) kinase kinase, and an agent that inhibits MAP kinase may be, but not limited to: N-[(2S)-2,3-dihydroxypropyl]-3-[ (2-fluoro-4-iodophenyl)amino]-4-pyridinecarboxamide ("AS-703026") (Kim, Kong et al., 2010), N-[(2R)-2,3-dihydroxypropyl ]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]benzamide ("PD0325901") (Thompson and Lyons, 2005), 5-(2-phenyl-pyridine Azolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-ylamine ("FR 180204") (Ohori, Kinoshita et al., 2005), 2-(2-Amino-3-methoxyphenyl)-4H-chromen-4-one ("PD98059") (Alessi, Cuenda et al., 1995), 6-(4-bromo-2-chlorophenyl Amino)-7-fluoro-N-(2-hydroxyethoxy)-3-methyl-3H-benzo[d]imidazole-5-carboxamide ("Selumetinib") (Huynh, Soo et al., 2007), (Z)-3-Amino-3-(4-aminophenylsulfanyl)-2-(2-(trifluoromethyl)phenyl)acrylonitrile ("SL-327") (Chen, Operana et al., 2005 ), (2Z,3Z)-2,3-bis(amino(2-aminophenylthio)methylene)succinonitrile, ethanol ("U0126") (Favata, Horiuchi et al., 1998), (R) -3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4 , 7(3H,8H)-diketone ("TAK-733") (Dong, Dougan et al., 2011) and N-(3-(3-cyclopropyl-5-(2-fluoro-4-iodophenyl Amino)-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1-(2H)-yl)benzene base) acetamide ("Trametinib") (Gilmartin, Bleam et al., 2011). Reagents that inhibit RTK can be, but are not limited to: N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinone Azolinamine ("Gefitinib") (Ciardiello 2000), (E)-2-cyano-3-(3,4-dihydroxyphenyl)-2-acrylamide ("AG 99" )(Gazit, Yaish et al., 1989), 4-[[(2S)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]-3-[7-methyl-5-(4- Morpholinyl)-1H-benzimidazol-2-yl]-2(1H)-pyridone ("BMS536924") (Huang, Greer et al., 2009), 5-(2-phenyl-pyrazolo[1 ,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-ol ("FR 180209") (Anastassiadis, Duong-Ly et al., 2013), N-( 3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine hydrochloride ("Erlotinib") (Kuiper, Heideman et al., 2014), (S,E)-N-(4-(3-chloro-4-fluorophenylamino)-7-(tetrahydrofuran-3-yloxy)quinazolin-6-yl)-4-( Dimethylamino)but-2-enamide ("Afatinib") (Minkovsky and Berezov, 2008), N-(4-(3-fluorobenzyloxy)-3-chlorophenyl) -6-(5-((2-(methylsulfonyl)ethylamino)methyl)furan-2-yl)quinazolin-4-amine, bis(4-methylbenzenesulfonate)(" Lapatinib (Lapatinib)")(Xia, Mullin et al., 2002), N-(3-(5-chloro-2-(2-methoxy-4-(4-methylpiperazin-1-yl )phenylamino)pyrimidin-4-yloxy)phenyl)acrylamide ("WZ4002") (Sakuma, Yamazaki et al., 2012) and 2-[(3,4-dihydroxyphenyl)methylene]- ("AG-18", malononitrile) (Gazit, Yaish et al., 1989).

The cell culture solutions and systems of the invention may additionally be used to form three-dimensional tissue constructs comprising transplantable epithelium for regenerative purposes. Such tissue constructs can be grafted into the host according to methods known in the art (Lloyd et al., 2006; Gupta et al., 2006; Yui et al., 2012). Tissues susceptible to treatment include all damaged tissues, including those that may have been damaged by disease, injury, trauma, autoimmune response, or by viral or bacterial infection. Minimally invasive grafting techniques, including image-guided techniques, can be used. Tissue constructs can be injected or implanted directly into damaged tissue where they can replicate and eventually differentiate into desired cell types depending on their location in the body. Tissue constructs can be directly implanted or infused via a colonic enema. For upper intestinal applications, micronization may be employed prior to oral delivery. Therefore, damaged tissues particularly suitable for repair include the colon, small intestine, pancreas, esophagus and gastric system. The skilled artisan will understand what the appropriate dosage of the tissue construct is for the particular condition being treated.

The cell culture solutions and systems of the invention can additionally be used to predict the efficacy of chemotherapeutic agents or combinations of chemotherapeutic agents in vivo. Such approaches are particularly relevant for use in clinical settings where many patients are treated with multiple drugs.

By culturing isolated tumor cell aggregates or single cells in the culture solution of the present invention, tumor organoids can be formed according to methods known in the art (Sato et al., 2011a). Such cultures can be used as clinical models for various cancers including, but not limited to, prostate, breast, stomach, pancreas, lung, brain, colon, small intestine, and bladder.

Tumor organoids can be incubated in the presence of cell culture solutions of the invention (e.g., including BMP inhibitors, R-spondin 1, Wnt agonists, histone deacetylase inhibitors) and chemotherapeutic agents. Subsequently, the relevant parameters are determined and evaluated. Relevant parameters include inhibition of cell viability, inhibition of cell proliferation, inhibition of tumor-associated gene expression, activation of apoptosis, and inhibition of cell survival. Detecting an increase in a parameter compared to a reference (e.g., control) indicates the efficacy of the chemotherapeutic agent relative to the tumor organoid, which is predictive of the efficacy of the chemotherapeutic agent in vivo.

In general, chemotherapeutic agents are incubated with the cell culture system at doses estimated to be therapeutic and for a duration sufficient to produce a physiological effect. The incubation time can be from about 1 hour to about 24 hours, or can be extended to days or even weeks as desired. Incubation conditions generally involve using the culture solution of the present invention and maintaining a temperature of about 37°C.

A chemotherapeutic agent is any substance that has been evaluated for its ability to cure, alleviate, treat or prevent cancer in a subject and may include, but is not limited to, chemical compounds, biological agents, proteins, peptides, nucleic acids, lipids, polysaccharides , supplements and antibodies.

Inhibition of expression of tumor-associated genes can be determined according to methods known in the art. For example, inhibition of tumor-associated gene expression relative to controls can be detected by microchip analysis, RT-PCR, in situ hybridization, fluorescence in situ hybridization, or Northern analysis. Inhibition of tumor-associated protein expression relative to controls can be detected by quantitative Western blot, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay, amino acid sequence analysis, fluorescence-activated cell sorting, or protein concentration testing. For example, gastric cancer genetic screening tests can be used to identify angiotensin, apolipoprotein E, apolipoprotein A-I, ceruloplasmin, prothrombin, fibronectin, vitamin D binding protein, gelsolin, inter-alpha trypsin inhibitor Changes in gene expression of heavy chain H3, kininogen-1, serum paraoxonase/aromatic esterase 1, alpha-1-antichymotrypsin, and transthyretin.

Activation of apoptosis can be determined according to methods known in the art. For example, an increase in cell death relative to a control can be detected by lactate dehydrogenase release, caspase activity, annexin V staining, phosphatidylserine staining, or TUNEL assay. Certain tests detect relatively late events in the cell death process, such as the release of lactate dehydrogenase. Caspase activation is a shared feature of chronic toxicity and cell death. Caspase activity can be measured relatively quickly (30 minutes to 4 hours) after toxic insult by fluorescence spectroscopy, thus lending itself to high-throughput screening techniques. Other markers and tests commonly used to detect apoptosis or necrosis of cells may include, but are not limited to, the presence of phosphatidylserine on the outer layer of the plasma membrane of affected cells, Annexin V staining, and terminal deoxynucleotidyl transferase Nick-end labeling test (TUNEL).

Inhibition of cell viability can be determined according to methods known in the art including, but not limited to, the use of vital dyes such as trypan blue, 4,6-diaminophenylindole (DAPI), and pyridinium iodide for viability Differential counts of cells and dead cells.

Inhibition of cell proliferation can be determined according to methods known in the art including, but not limited to, DNA quantification by bromodeoxyuridine incorporation, measurement of tritiated thymidine (3H-thymidine), pyridium iodide staining, staining by Intracellular metabolic analysis of tetrazolium salt or AlamarBlue reduction and quantification of intracellular ATP concentration. Other methods include: measuring the total nucleic acid content of lysed cells by spectrophotometric analysis; using anti-cdc6 peptide antibodies, anti-human mRNA binding protein HuR antibodies (anti-HuR antibodies), antibodies against D cyclins and cyclin-dependent kinase inhibitors Fluorescent labeling of antibodies; Ki-67 antigen detection; determination of protein content by quantitative Western blotting, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay, amino acid sequence analysis, fluorescence-activated cell sorting, or protein concentration testing. Commercially available kits using the methods described above include: ChromaTide ^™ Nucleotide Labeling, Succinimidyl Ester of Carboxyfluorescein Diacetate, ABSOLUTE-S ^™ SBIP Cell Proliferation Assay Kit, Vybrant DiI Cell Labeling Solution, CyQUANT Cell Proliferation Assay Kit, Vybrant ^TM MTT Cell Proliferation Assay Kit and FluoReporter ^TM Blue Fluorescence Analysis Nucleic Acid Test Kit.

Suppression of cell survival can be determined according to methods known in the art including clonogenic assays.

III. Methods of Promoting Epithelial Cell Expansion or Epithelial Tissue Growth in Vivo

Epithelial stem cells, including those of the intestine, stomach, lung, pancreas, and colon, can be expanded in vivo by administering a Wnt agonist and a histone deacetylase inhibitor or a Wnt agonist and a Notch agonist to a subject. Stem cells, especially LGR5-positive stem cells present in the small intestine, inner ear, brain, kidney, liver, retina, stomach, pancreas, breast, hair follicles, ovary, adrenal medulla, skin, thymus, taste buds, and mammary glands. These combinations promote the expansion of epithelial cells, resulting in the growth of epithelial tissue in vivo.

In specific embodiments, a Wnt agonist (eg, CHIR99021) and a histone deacetylase inhibitor (eg, valproic acid) or a Wnt agonist (eg, CHIR99021 ) and a Notch agonist may be administered to a subject. Enterocytes are then formed in vivo.

In certain embodiments, these combinations (eg, CHIR99021 and valproic acid) can treat intestinal disorders in a subject, including but not limited to: enterocolitis; viral infections, such as non-specific enteritis or specific viral enteritis; diverticulitis; bacterial enterocolitis such as salmonellosis, shigellosis, Campylobacter enterocolitis, or Yersinia enterocolitis; protozoan infection such as amoebiasis; helminth infection (helminthic infection); and pseudomembranous colitis, and pulmonary complications of chronic obstructive pulmonary disease and cystic fibrosis; appendicitis; atrophic gastritis; Barrett's esophagus disease; pneumonia; cervicitis; chronic interstitial nephritis; colitis ; diverticulitis of the colon; conjunctivitis; contact dermatitis; Colin's ulcer; Cushing's ulcer; cystitis; gangrene; gingivitis; mastitis; esophagitis; pancreatitis; panniculitis; cellulitis gastritis; kidney glomerulonephritis; and font immune diseases, which include, but are not limited to, inflammatory bowel disease, ulcerative colitis, Crohn's disease, Addison's disease, and glomerulonephritis (e.g., crescentic glomerulonephritis, proliferative glomerulonephritis).

The dose administered will depend on the age, sex, health and weight of the recipient, the type (if any) of concurrent treatment, the frequency of treatment and the nature of the effect desired. The compositions of the invention are administered in a dosage range that is sufficient to produce the desired effect. The dose should not be so large as to cause adverse side effects, such as unwanted cross-reactions, allergic reactions, and the like. In general, dosage will vary with the condition and extent of disease of the patient. Counterindications (if any), immune tolerance, and other variables will also affect appropriate dosages. For example, factors such as the patient's age, weight, sex, species, general health/condition, condition to be treated, timing of treatment, LD50 of the active ingredient involved in a suitable animal model (e.g., rodent, mouse) and Other known factors and the like; and such doses may be in the order of milligrams (for example, in the order of 0.5 mg/kg to 500 mg/kg) or other suitable amounts, or may be calculated from the examples herein, for example considering to the average body weight of a typical subject animal (eg, mouse) and the dose administered thereto (eg, 100 mg), and thus can be determined by one skilled in the art without undue experimentation. Specifically, in human subjects, CHIR99021 was administered at an amount of about 0.1 mg/kg/day to about 100 mg/kg/day, while valproic acid was administered at an amount of about 1 mg/kg/day to about 1000 mg/kg/day. The daily amount is administered. In a specific embodiment, the amount of valproic acid is 15 mg/kg/day to about 40 mg/kg/day.

The pharmaceutical composition of CHIR99021 and valproic acid can be administered simultaneously or sequentially by any means that achieve their intended purpose. For example, administration can be topical, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, rectal, or buccal. Alternatively, or simultaneously, administration may be by the oral route. From the above description it will be apparent that the invention described herein can be varied and modified to adapt it to various applications and conditions. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are presented for illustrative purposes only and not intended to be limiting. Such implementations also fall within the scope of the following claims. The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation herein of an embodiment includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof. Any publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated by reference in their entirety.

Exemplary embodiments herein may also be described by any of the following numbered paragraphs:

1. A method of forming intestinal epithelial cells in a cell culture system, said method comprising culturing epithelial stem cells in the presence of at least one Wnt inhibitor and at least one histone deacetylase inhibitor, said at least A Wnt inhibitor and at least one histone deacetylase inhibitor are each in an amount sufficient to generate intestinal epithelial cells in a cell culture system.

2. The method of paragraph 1, wherein the histone deacetylase inhibitor is a Pan-HDAC inhibitor.

3. The method of paragraph 2, wherein the Pan-HDAC inhibitor is selected from the group consisting of valproic acid, trichostatin A, suberoylanilide hydroxamic acid and SBHA.

4. The method of paragraph 1, wherein the histone deacetylase inhibitor is an HDAC6 inhibitor.

5. The method of paragraph 4, wherein the HDAC6 inhibitor is selected from the group consisting of Tubastatin, Tubastatin A and Compound 7.

6. The method of paragraph 1, wherein the Wnt agonist is selected from the group consisting of IWP-2, XAV-939, ICG-001, LGK-974, IWR-1-endo, KY02111, Wnt-C59, DKK-1 , FH-535, Box5, peptide Pen-N3, anti-SFRP antibody and anti-LRP6 antibody.

7. The method of paragraph 1, further comprising incubating the epithelial stem cells in the presence of an inhibitor of bone morphogenetic protein.

8. The method of paragraph 7, wherein the inhibitor of bone morphogenetic protein is selected from the group consisting of noggin, chordrin, follistatin, DAN, proteins containing DAN cysteine knot domains, bone Group consisting of sclerostin, gastrulation protein, uterine sensitivity-related gene-1, connective tissue growth factor, inhibin, BMP-3, and dorsomorphin.

9. The method of paragraph 1, further comprising incubating the epidermal stem cells in the presence of epidermal growth factor.

10. A method of forming goblet cells in a cell culture system, said method comprising incubating epithelial stem cells in the presence of at least one Wnt inhibitor and at least one Notch inhibitor, said at least one Wnt inhibitor and at least one Notch inhibitor are each in an amount sufficient to generate goblet cells in a cell culture system.

11. The method of paragraph 10, wherein the Notch inhibitor is selected from the group consisting of DAPT, RO4929097, LY450139, LY900009, LY3039478, LY411575, YO-01027, BMS-708163, BMS-906024, Compound E, BMS-299897, Panel consisting of SAHM1, Abeta42-Selective and SB 225002.

12. The method of paragraph 10, wherein the Wnt inhibitor is selected from the group consisting of IWP-2, XAV-939, ICG-001, LGK-974, IWR-1-endo, KY02111, Wnt-C59, DKK-1 , FH-535, Box5, peptide Pen-N3, anti-SFRP antibody, anti-LRP6 antibody and anti-APC antibody.

13. The method of paragraph 10, further comprising incubating the epidermal stem cells in the presence of epidermal growth factor.

14. A method for forming enteroendocrine cells in a culture system, the method comprising inhibiting receptor tyrosine kinases, mitogen-activated protein (MAP) kinases, or extracellular signal-regulated kinases ( The epithelial stem cells are incubated in the presence of at least one agent of ERK), the amount of each of the at least one Notch inhibitor and the agent is an amount sufficient to generate enteroendocrine cells in a cell culture system.

15. The method of paragraph 14, wherein the Notch inhibitor is selected from the group consisting of DAPT, RO4929097, LY450139, LY900009, LY3039478, LY411575, YO-01027, BMS-708163, BMS-906024, Compound E, BMS-299897, Panel consisting of SAHM1, selective Abeta42 and SB 225002.

16. The method of paragraph 14, wherein the MAP kinase is mitogen-activated protein kinase kinase (MEK).

17. The method of paragraph 14, wherein the agent inhibiting MAP kinase is selected from the group consisting of AS-703026, PD0325901, PD98059, Selumetinib, SL-327, U0126, TAK-733 and Trametinib ( Trametinib) group.

18. The method of paragraph 14, wherein the agent that inhibits RTK is selected from the group consisting of Gefitinib, AG99, Erlotinib, Afatinib, Lapatinib ), WZ4002 and AG-18.

19. The method of paragraph 14, wherein the agent that inhibits ERK is AS-703026 or PD0325901.

20. The method of paragraph 14, further comprising incubating the epithelial stem cells in the presence of an inhibitor of bone morphogenetic protein.

21. The method of paragraph 20, wherein the bone morphogenetic protein is selected from the group consisting of noggin, chordrin, follistatin, DAN, DAN cysteine knot domain containing protein, sclerostin, Group consisting of gastrulation protein, uterine sensitivity-related gene-1, connective tissue growth factor, inhibin, BMP-3, and Dorsomorphin.

22. The method of paragraph 14, further comprising incubating the epithelial stem cells in the presence of an agent that binds to leucine-rich repeat G protein-coupled receptor 5.

23. The method of paragraph 22, wherein the agent that binds to leucine-rich repeat G protein-coupled receptor 5 is selected from the group consisting of R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin A group consisting of 4 spondin.

24. The method of paragraph 14, further comprising incubating the epidermal stem cells in the presence of epidermal growth factor.

25. A method of forming intestinal epithelial cells in a subject in need thereof, said method comprising administering to said subject at least one Wnt agonist in an amount sufficient to form intestinal epithelial cells in the subject and histone deacetylase inhibitors.

26. The method of paragraph 25, wherein the subject is a human.

27. The method of paragraph 25, wherein the Wnt agonist is from the group consisting of: Wnt-1/Int-1, Wnt-2/Irp (Int-1-related protein), Wnt-2b/13 , Wnt-3/Int-4, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a/8d, Wnt-8b, Wnt-9a /14, Wnt-9b/14b/15, Wnt-10a, Wnt-10b/12, Wnt-11, Wnt-16, R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, Nuo Liberin, CHIR99021, LiCl, BIO((2'Z,3'E)-6-bromoisatin-3'-oxime), CHIR98014, SB 216763, SB415286, 3F8, Kemparolone, 1-Aza Camparolone, TC-G24, TCS 2002, AR-A 014418, 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxybenzene base) pyrimidine, IQ 1, DCA, QS 11, WAY-316606, (hetero)aryl pyrimidine, 10Z-hemandex, TCS 21311, TWS 119, GSK-3 inhibitor IX, GSK-3 inhibitor IV, GSK -3β Inhibitor II, GSK-3β Inhibitor I, GSK-3β Inhibitor XXVII, GSK-3β Inhibitor XXVI, FRATtide, Cdk1/5 Inhibitor, Bikinin and 1-Azacamparolone.

28. The method of paragraph 25, wherein the histone deacetylase inhibitor is a Pan-HDAC inhibitor.

29. The method of paragraph 28, wherein the Pan-HDAC inhibitor is selected from the group consisting of valproic acid, trichostatin A, suberoylanilide hydroxamic acid and SBHA.

30. The method of paragraph 25, wherein the histone deacetylase inhibitor is an HDAC6 inhibitor.

31. The method of paragraph 30, wherein the HDAC6 inhibitor is selected from the group consisting of tupastatin, tubastatin A and compound 7.

32. The method of paragraph 25, wherein the Wnt agonist is CHIR99021 and the histone deacetylase inhibitor is valproic acid.

33. The method of paragraph 32, wherein the CHIR99021 is administered in an amount from about 0.1 mg/kg/day to about 100 mg/kg/day and valproic acid is administered in an amount from about 1 mg/kg/day to about 1000 mg/kg /daily dose.

34. A method of generating epithelial tissue in a subject in need thereof, said method comprising administering to said subject at least one Wnt agonist and a combination thereof sufficient to increase the amount of epithelial stem cells within said epithelial tissue A protein deacetylase inhibitor or a Wnt agonist and a Notch agonist, thereby generating epithelial tissue in said subject.

35. The method of paragraph 34, wherein said epithelial stem cells are present in the small intestine, inner ear, brain, kidney, liver, retina, stomach, pancreas, breast, hair follicle, ovary, adrenal medulla, skin, thymus, taste buds or LGR5-positive stem cells in the mammary gland.

36. A method of forming small intestinal epithelial cells in a test subject in need thereof, said method comprising administering to said test subject at least one Wnt agonist in an amount sufficient to form small intestinal epithelial cells in the test subject and Notch agonists.

35. The method of paragraph 34 or 36, wherein the subject is a human.

36. The method of paragraph 34 or 36, wherein the Wnt agonist is selected from the group consisting of: Wnt-1/Int-1, Wnt-2/Irp (Int-I-related protein), Wnt- 2b/13, Wnt-3/Int-4, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a/8d, Wnt-8b, Wnt-9a/14, Wnt-9b/14b/15, Wnt-10a, Wnt-10b/12, Wnt-11, Wnt-16, R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4. Norrie protein, CHIR99021, LiCl, BIO ((2'Z,3'E)-6-bromoisatin-3'-oxime), CHIR98014, SB 216763, SB415286, 3F8, camparone, 1 -Azacamparolone, TC-G24, TCS 2002, AR-A 014418, 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methyl Oxyphenyl)pyrimidine, IQ 1, DCA, QS 11, WAY-316606, (hetero)arylpyrimidine, 10Z-hemandex, TCS 21311, TWS 119, GSK-3 Inhibitor IX, GSK-3 Inhibitor IV, GSK-3β Inhibitor II, GSK-3β Inhibitor I, GSK-3β Inhibitor XXVII, GSK-3β Inhibitor XXVI, FRATtide, Cdk1/5 Inhibitor, Bikinin and 1-Azacamparolone.

37. The method of paragraph 34 or 36, wherein the Notch agonist is Notch 1 antibody (Nl Ab), Delta 1, Delta-like 3, Delta-like 4, Jagged 1, Jagged 2, DSL peptide, and Delta D.

38. A method of treating a small bowel disorder comprising administering to a subject a Wnt agonist and a histone deacetylase inhibitor or a Wnt agonist and Notch.

39. The method of paragraph 38, wherein the subject is a human.

40. The method of paragraph 38, wherein the Wnt agonist is selected from the group consisting of: Wnt-1/Int-1, Wnt-2/Irp (Int-I-related protein), Wnt-2b/ 13. Wnt-3/Int-4, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a/8d, Wnt-8b, Wnt- 9a/14, Wnt-9b/14b/15, Wnt-10a, Wnt-10b/12, Wnt-11, Wnt-16, R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, Norrie protein, CHIR99021, LiCl, BIO((2'Z,3'E)-6-bromoisatin-3'-oxime), CHIR98014, SB 216763, SB415286, 3F8, ceparolone, 1-nitrogen Heterocaprolone, TC-G24, TCS 2002, AR-A 014418, 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxy Phenyl)pyrimidine, IQ 1, DCA, QS 11, WAY-316606, (hetero)arylpyrimidine, 10Z-hemandex, TCS 21311, TWS 119, GSK-3 Inhibitor IX, GSK-3 Inhibitor IV, GSK-3β Inhibitor II, GSK-3β Inhibitor I, GSK-3β Inhibitor XXVII, GSK-3β Inhibitor XXVI, FRATtide, Cdk1/5 Inhibitor, Bikinin and 1-Azacamparolone.

41. The method of paragraph 38, wherein the histone deacetylase inhibitor is a Pan-HDAC inhibitor.

42. The method of paragraph 41, wherein the Pan-HDAC inhibitor is selected from the group consisting of valproic acid, trichostatin A, suberoylanilide hydroxamic acid, and SBHA.

43. The method of paragraph 38, wherein the histone deacetylase inhibitor is an HDAC6 inhibitor.

44. The method of paragraph 43, wherein the HDAC6 inhibitor is selected from the group consisting of tupastatin, tubastatin A and compound 7.

45. The method of paragraph 38, wherein the Wnt agonist is CHIR99021 and the histone deacetylase inhibitor is valproic acid.

46. The method of 45, wherein said CHIR99021 is administered in an amount of about 0.1 mg/kg/day to about 100 mg/kg/day, and valproic acid is administered at about 1 mg/kg/day to about 1000 mg/kg/day amount applied.

47. The method of paragraph 38, wherein the Notch agonist is Notch 1 antibody (Nl Ab), Delta 1, Delta-like 3, Delta-like 4, Jagged 1, Jagged 2, DSL peptide, and Delta D.

48. The method of any one of paragraphs 38 to 47, wherein the small bowel disorder is selected from the group consisting of: enterocolitis; viral infection, such as nonspecific enteritis or specific viral enteritis; diverticulitis; Bacterial enterocolitis, such as salmonellosis, shigellosis, Campylobacter enterocolitis, or Yersinia enterocolitis; protozoan infections, such as amoebiasis; helminth infections; and pseudomembranous colitis and pulmonary complications of cystic fibrosis and chronic obstructive pulmonary disease; appendicitis; atrophic gastritis; Barrett's esophagus; pneumonia; cervicitis; chronic interstitial nephritis; colitis; diverticulitis of the colon; conjunctivitis; Contact dermatitis; Colin's ulcer; Cushing's ulcer; cystitis; gangrene; gingivitis; mastitis; esophagitis; pancreatitis; panniculitis; cellulitic gastritis; glomerulonephritis; and autoimmune diseases , including but not limited to inflammatory bowel disease, ulcerative colitis, Crohn's disease, Addison's disease, and glomerulonephritis (eg, crescentic glomerulonephritis, proliferative glomerulonephritis).

The invention is further described in the following examples, but these examples do not limit the scope of the invention described in the claims.

Example

Example 1. Self-renewal of Lgr5 ⁺ small intestinal stem cells is maintained using a combination of small molecules

Self-renewal and differentiation of ISCs is controlled through the coordinated regulation of several signaling pathways (Crosnier, Stamataki, & Lewis, 2006; Scoville, Sato, He, & Li, 2008; van der Flier & Clevers, 2009). In this study, small molecules were identified that target relevant signaling pathways to maintain the self-renewal state of Lgr5 ⁺ stem cells ^and control them independently of cues provided by other cell types differentiation.

Crypts and single Lgr5-GFP cells were isolated as previously described (Sato et al., 2009). Briefly, the proximal half of the small intestine was harvested, opened longitudinally and washed with cold PBS to remove luminal contents. The tissue was then cut into 2 to 4 mm pieces with scissors, and washed 5 to 10 times with cold PBS by pipetting out with a 10-ml pipette. Tissue fragments were incubated with 2 mM EDTA in PBS for 30 minutes on ice. After removal of EDTA, the tissue fragments were washed with PBS to release the crypts. The crypt-enriched supernatant fraction was collected, passed through a 70 μm cell strainer and centrifuged at 300 g for 5 minutes. Cell pellets were resuspended in cell culture medium without growth factors and centrifuged at 150 g to remove single cells. The crypts are then cultured or used for single cell isolation. To obtain single cells, crypts were incubated in medium for 45 minutes at 37°C and then broken up with a glass pipette. Dissociated cells were passed through a 20 μm cell strainer, which were negatively stained with propidium iodide, and single viable high GFP cells. Intestinal crypts isolated from Lgr5-EGFP-ires-CreERT2 mice were embedded in Matrigel and cultured under conventional culture conditions in the presence of EGF, noggin and R-spondin 1 (collectively referred to as ENR), thereby Organoids with crypts and villus-like domains and GFP ⁺ cells at the crypt tips were generated, consistent with previous reports (Sato et al., 2009). Isolated crypts or single cells were cultured as previously described (Sato et al., 2009) with minimal modifications. Briefly, crypts or single cells were embedded in Matrigel and plated into the centers of wells of 24-well plates. After polymerization of Matrigel (growth factor-reduced; BD Bioscience), 500 μl of medium (Advanced DMEM/F12 (Life Technologies)) containing growth factors and small molecules including EGF (50 ng/ml, Life Technologies) was added , noggin (100ng/ml, Peprotech) and R-spondin 1 (500ng/ml, R&D), small molecules include CHIR99021 (3μΜ, Stemgent) and valproic acid (1mM, Sigma-Aldrich). To compare different culture conditions, small molecules or growth factors were added to freshly isolated crypts immediately after plating in Matrigel in order to test the ability to minimize the potential differentiation of ISCs within the crypts and thereby maintain crypt cultures. ability. Change the cell culture medium every other day. For single-cell cultures, cells were embedded in Matrigel containing Jagged-1 peptide (1 μΜ; AnaSpec) and Y-27632 (10 μΜ; Tocris) was added for the first 2 days. Cells were passaged as cell colonies as previously described (Sato et al., 2009) or as single cells. For single cell passaging, cell culture medium was removed and Accutase (Life Technologies) was added. After incubation at 37°C for 10 to 20 minutes, cell colonies were dissociated into single cells by pipetting. Cells were then washed, embedded in fresh Matrigel and plated into 24-well plates. Cells cultured under CV conditions were passaged every 6 days at a split ratio of 1:20. Approximately half of the cultured crypts contained GFP+ cells, consistent with in vivo GFP expression in Lgr5-GFP mice (Fig. 1).

The growth factors used in ENR conditions provided critical but insufficient cues to maintain self-renewal of Lgr5 ⁺ stem cells. To identify key factors that maintain the self-renewal state of intestinal stem cells, selected small molecules that modulate ISC signaling pathways, such as Wnt, Notch, and BMP, were tested under ENR conditions using the Lgr5-GFP reporter. CHIR99021 (referred to herein as CHIR or C) is a GSK3 inhibitor that activates the Wnt signaling pathway, which promotes the proliferation of crypt cells, as indicated by the quantification of the average size and cell number of organoids in culture ( Figures 2A, 2B and 3A, 3B). CHIR increased the percentage of GFP ⁺ cells in culture and the relative GFP intensity, indicative of increased stem cell self-renewal (Figures 2A and 2B). In particular, a large number of GFP-negative cells remained within the organoids (Fig. 2A), which may be the result of insufficient maintenance of stem cell self-renewal or the promotion of proliferation of more mature GFP-negative cells in the crypts. Valproic acid (VPA or V), a histone deacetylase inhibitor, also significantly increased GFP expression in GFP ⁺ organoids in the presence of minimal GFP-negative cells (Fig. 2A). Interestingly, when CHIR and VPA were combined (CV), cell proliferation in culture was significantly increased as well as the percentage of cells expressing GFP and the relative GFP intensity (Figures 2A and 2B), and GFP ⁺ organoids In , there were nearly pure GFP+ cells (Fig. 2A), suggesting that differentiation or proliferation of differentiated cells was minimized and stem cell self-renewal was increased in this culture condition.

GFP ⁺ cells under CV conditions showed a single high GFP population corresponding to freshly isolated single cells (Fig. 3C), exhibiting a Lgr5 ⁺ stem cell population as previously reported (Sato et al., 2009). In particular, under CV conditions, R-spondin 1 and noggin are still required to maintain the self-renewal of Lgr5 ⁺ stem cells, while EGF promotes the proliferation of crypts, which can be removed from culture without affecting the ability of Lgr5+ stem cells. Maintenance of ⁺ cells (Fig. 3D). Increasing CHIR concentrations further abolished the requirement for R-Spondin 1 to promote GFP expression (Fig. 3E), consistent with the role of R-Spondin 1 in increasing Wnt/β-catenin signaling. In addition, VPA or CHIR+VPA also promoted GFP expression in colon-derived Lgr5+ stem cells (Fig. 3F). In addition, compared with R-Spondin 1, R-Spondin 2 showed better potency at lower concentrations in promoting organoid formation under ENR conditions (Fig. 3G). The inventors also tested culture conditions previously shown to maintain human EPHB2 ⁺ colonic stem cells or colonic crypts in a largely undifferentiated state (Jung et al., 2011; Sato et al., 2011a), but failed for small intestinal Lgr5-GFP stem cells. Similar effects were achieved (Figures 4A and 4B), suggesting that these factors may act through different mechanisms on EPHB2 ⁺ colon stem cells versus Lgr5 ⁺ stem cells.

To further confirm the proliferative and Lgr5 ⁺ self-renewal effects of CHIR and VPA in the absence of mature cell types and GFP-negative stem cells (provided that crypts displayed a mosaic GFP expression pattern), single high GFP cells were isolated by FACS sorting (FIG. 3C) and cultured in Matrigel in the presence of ENR and either CHIR or VPA or both compounds (CV conditions). The Rho kinase inhibitor Y-27632 (Watanabe et al., 2007), which inhibits anoikis of individual stem cells, was added for the first two days as previously described (Sato et al., 2009). Colonies containing GFP ⁺ stem cells formed spontaneously after 7 days in culture. Similar to crypt cultures, CHIR significantly increased cell proliferation but it only moderately increased GFP expression, while VPA promoted GFP expression with minimal pro-proliferative effects. For CV conditions, cell proliferation was significantly increased, and >97% of the cells in the medium were GFP ⁺ cells (Figures 2C-2E and 5A). Notably, when pure single Lgr5 ⁺ stem cells were cultured in CHIR compared to crypt cultures, the organoids formed contained a large number of GFP-negative cells, thus suggesting that stem cells differentiate under this condition and therefore require other factors to maintain the self-renewal state of Lgr5 ⁺ stem cells.

When single Lgr5-GFP ⁺ cells were cultured under standard ENR conditions, very few cells grew into organoids, consistent with previous reports (Sato et al., 2009), and most likely due to suboptimal culture conditions. When CHIR was added to the culture (ENR-C), the colony formation efficiency was significantly increased by 20-fold to 50-fold (Fig. 2F, 2G and 5B, 5C), providing a similar response to Wnt3A when added at 100 ng/ml (Fig. 2F and Sato et al., 2011b). In sharp contrast, VPA only weakly increased colony formation efficiency in the absence of CHIR (ENR-V, Figures 2F, 2G and 5B, 5C). Surprisingly, when isolated single Lgr5-GFP ⁺ stem cells were cultured in the presence of both CHIR and VPA, there was a synergistic effect and approximately 25% to 40% of the total cell population grew as colonies (Fig. 2F). This is thought to represent the most efficient colony formation ever reported for Lgr5 ⁺ stem cells.

Table 1. Number of colonies formed by the colonies in Figure 2G

ENR ENR--C ENR-V ENR-CV ENR-W ENR-WV average 7.333333 158.6667 32.33333 956 135.3333 475.3333

Table 2. Colony Numbers for Colony Formation Efficiency in Figure 5C

ENR ENR-C ENR-W ENR-V ENR-CV average 3 164.75 56.25 24.5 495.25

Considering that a fraction of FACS-sorted cells are in a pro-apoptotic state and typically die within 12 hours (Sato et al., 2011b), live cells were counted manually 12 hours after seeding. When both CHIR and VPA were present in the medium, greater than 90% of viable cells grew into organoids (Fig. 5D).

Table 3. Colony formation efficiency in Figure 5D

ENR ENR-V ENR-C ENR-CV ENR-W ENR-WV average 0 0 0.142291 0.921154 0.132576 0.190111

Note: <100 cells were seeded, so calculated efficiency is 0 for R or RV

Furthermore, cells cultured under CV conditions could be passaged as single cells for more than 10 passages with similar colony-forming efficiency as freshly isolated Lgr5-GFP ⁺ cells without loss of proliferative ability, and they displayed a normal karyotype (2n = 40) (Fig. 2H). These results suggest that CHIR and VPA provide signals absent in standard ENR conditions to maintain self-renewal of Lgr5 ⁺ stem cells.

As previously reported, cells under ENR conditions grew into organoids with crypt-villus architecture containing all small intestinal epithelial cell types, which consisted of alkaline phosphatase (Alp)-positive intestinal epithelial cells, mucin 2 (Muc2)-positive This was confirmed by staining of goblet cells, chromogranin A (ChgA)-positive enteroendocrine cells, lysozyme (Lyz)-positive Paneth cells, and Lgr5-GFP ⁺ stem cells. Lgr5 ⁺ stem cells reside only at the tips of the crypts (Figures 6A and 7A). Ki67 and EdU staining revealed that proliferating cells were only present within the crypt domain (Figures 6B and 6C). However, under CV conditions, GFP ⁺ stem cells were present throughout the colonies with very few Paneth cells (Fig. 6A) and no other cell types. Ki67- or EdU-positive proliferating cells under CV conditions were present throughout the cell colonies compared to ENR cultures (Figures 6B and 6C). This was confirmed by quantitative real-time PCR that cells under CV conditions expressed minimal levels of Alpi (intestinal epithelial cells), Muc2 (goblet cells), ChgA (enteroendocrine cells), intermediate levels of lysozyme (Paneth cells) and high levels of Lgr5 (ISC) (Fig. 6D). This expression pattern was maintained over multiple passages, as was the level of Lgr5 expression (Fig. 6D).

CHIR alone decreased intestinal epithelial cell differentiation but simultaneously increased Paneth cell differentiation (Fig. 6D), which is consistent with previous reports (Farin et al., 2012). While VPA alone reduced secretory differentiation (Fig. 6D) and helped maintain a higher proportion of GFP+ stem cells, it was not sufficient to inhibit stem cell differentiation. In fact, when isolated single stem cells were cultured in the presence of VPA but not CHIR or other agents that promote Wnt signaling, their survival was much lower than when Wnt was present. When the Wnt pathway was blocked by IWP-2, VPA alone could not maintain the self-renewal of stem cells (IV condition in Fig. 7B, 7C). The combination of CHIR and VPA inhibited intestinal epithelial and secretory differentiation and maintained the self-renewal program of Lgr5 ⁺ stem cells (Fig. 6D). These results suggest that CHIR or VPA alone are not sufficient to maintain self-renewal of Lgr5 ⁺ stem cells, but show a synergistic effect when combined with CHIR or other Wnt activators.

Taken together, two small molecules, CHIR and VPA, can support the self-renewal of Lgr5 ⁺ stem cells without direct contact with Paneth cells or the absence of Paneth cells. In particular, these small molecules greatly improved colony formation from single stem cells, suggesting that they provide critical niche signals normally provided by Paneth cells.

Example 2. Lgr5 ⁺ stem cells maintain pluripotency after culture in CHIR and VPA

Intestinal stem cells have the ability to self-renew and differentiate into all cell types in the intestinal epithelium, including the four major cell types: enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. To test the differentiation capacity of Lgr5 ⁺ stem cells cultured under CV conditions, cell colonies were transferred to ENR conditions that allow Lgr5 ⁺ stem cells to spontaneously differentiate into mature cell types in the small intestine. As expected, after removal of CHIR and VPA, the morphology of the organoids changed to that typical of organoids cultured under ENR conditions, with a crypt-villi structure and Lgr5 ⁺ stem cells at the tips of the crypts (Fig. 7A and 8A). mRNA expression of differentiation markers Alpi, Muc2 and ChgA was increased and cells expressed similar levels of lysozyme (compare ENR and CV in Figure 7B).

Immunocytochemical staining for these markers confirmed the presence of differentiated cell types in culture (Fig. 7A).

Example 3. The differentiation of small intestinal stem cells is controlled

Next, with the ability to expand highly pure Lgr5+ stem cells in vitro, we attempted to induce differentiation of Lgr5+ stem cells into mature cell types. Since Wnt and Notch are the two main signaling pathways controlling ISC differentiation, the Wnt pathway inhibitor IWP-2 (also called I) and the Notch pathway inhibitor DAPT (also called D) were used to induce the differentiation of cultured Lgr5+ stem cells. As cells spontaneously differentiated into organoids containing all epithelial cell types under ENR conditions, ENR was included in the differentiation cultures. After culturing single stem cells under CV conditions for 6 days, cell colonies were harvested and transferred into wells and cultured in the presence of single or multiple inhibitors (Fig. 8B). As shown in Figure 7B, replacing CV with IWP-2 or DAPT reduced the expression of the ISC marker Lgr5 and induced the expression of the differentiation markers Alpi, Muc2, ChgA and lysozyme. In particular, the presence of VPA (for example, comparing R and V, I and IV, C and CV, or D and DV) resulted in lower levels of expression of Muc2, ChgA and lysozyme, but not Alpi, suggesting that VPA specifically inhibits the differentiation of secretory cell lines. Alternatively, Wnt inhibition with IWP-2 limitedly induced Alpi expression and moderately increased Muc2 and ChgA expression but completely abolished lysozyme and Lgr5 expression. This suggests that Wnt signaling is required to maintain sternness and inhibit differentiation, but is also required for Paneth cell differentiation. The Notch inhibitor DAPT greatly increased markers of secretory cell types including Muc2, ChgA, and lysozyme, consistent with previous reports that Notch inhibition induced differentiation of secretory cells (Milano et al., 2004; VanDussen et al. , 2012; Wong et al., 2004). Furthermore, the combination of IWP-2 and VPA presumably specifically induced intestinal epithelial differentiation by combining the effects of the two inhibitors, where IWP-2 induced differentiation of Lgr5 ⁺ stem cells while VPA inhibited the differentiation of Lgr5 ⁺ stem cells into secretory cell types. differentiation. Similarly, the combination of DAPT and CHIR mainly induced Paneth cell differentiation, whereas the combination of IWP-2 and DAPT mainly induced goblet cell differentiation. These conditions also induced distinct morphological changes resembling those of each differentiated cell type (Figures 7C and 8D). Staining for markers of intestinal epithelial cells, goblet cells and Paneth cells confirmed the above observations (Fig. 7C, 7D and 8E, 8F). ChgA expression was not significantly affected by the presence of IWP-2 or CHIR, suggesting that Wnt inhibition or activation is not strictly required for differentiation of enteroendocrine cells compared with goblet and Paneth cells.

Example 4. Examination of the Mechanisms Mediating the Responses of CHIR and VPA

CHIR is a highly specific GSK3 inhibitor that activates the Wnt/β-catenin signaling pathway (Bain et al., 2007) and has been used to maintain the self-renewal state of embryonic stem cells (Ying et al., 2008). To confirm that the effect of CHIR is achieved through activation of the Wnt pathway, the effects of other Wnt pathway activators including lithium and Wnt3a were tested. Replacing CHIR with LiCl or Wnt3a increased crypt proliferation, as indicated by an increase in colony size and cell number compared to ENR conditions (Figures 9A and 9B). Colonies under these conditions displayed cyst-like structures (Fig. 9A) as described previously (Sato et al., 2011b). Similarly, the effects of other HDAC inhibitors including pan-HDAC inhibitors and type-specific inhibitors were tested. The pan-HDAC inhibitor TSA, as well as the HDAC6-specific inhibitor Tubastatin A and compound 7 showed the effect of promoting GFP expression similar to that of VPA (Fig. 9C and 9D). However, HDAC inhibitors including SBHA and butyrate and class I (CI-994, MS275, Figure 9C and 9D), class IIA (MC1568, Figure 9C and 9D) and class III (nicotinamide, Figure 9F) Other pan-HDAC inhibitors showed no or only moderate effects in promoting GFP expression. TSA and VPA showed significant proliferation inhibitory effects at higher concentrations, but maintained GFP expression at both concentrations (Fig. 9E). Notably, nicotinamide, a sirtuin family HDAC inhibitor (class III), used in cultures of human colonic crypts (Jung et al., 2011; Sato et al., 2011a) did not promote GFP expression when combined with CHIR or Wnt3a or cell proliferation, suggesting that it acts through a mechanism different from that of VPA. Furthermore, when individual Lgr5 ⁺ stem cells were cultured using CHIR with TSA or tubastatin A, or VPA with Wnt3a, BIO, or LiCl, the cells displayed similar colony formation efficiency, colony morphology, and GFP expression to CV conditions (Fig. 10) .

Previous reports have shown that Notch pathway activation is required to inhibit secretory cell differentiation and maintain stem cell self-renewal, consistent with the effect of VPA treatment. Whether VPA targets elements of the Notch pathway to exert its effects was assessed. First, restoration of Notch inhibition by addition of VPA was tested, treatment with the gamma-secretase inhibitor DAPT resulted in impaired cell proliferation and GFP expression, which were restored by VPA in a dose-dependent manner (Fig. 11A). This suggests that VPA acts downstream of NICD formation and is able to circumvent ligand-receptor mediated Notch activation.

It was previously shown that VPA activates the Notch pathway in cancer cell lines (Greenblatt et al., 2007; Stockhausen et al., 2005). In order to study the effect of VPA on Notch pathway activation, cells cultured under ENR or ENRC conditions were treated with VPA, and the expression of Notch pathway genes was analyzed. However, it was determined that addition of VPA to ENR or ENR-C for 24 hours moderately reduced the expression of Notch1 or Hes1 (downstream target genes of Notch) (Figures 1 IB and 11C). In addition, a significant reduction of the negative Notch target Atoh1 (Math1 ) was observed in cells treated with VPA and CHIR for 24 hours or 6 days (FIGS. 11B-11D). Atoh1 has been shown to be critical for the differentiation of ISCs towards secreting lineages (van Es et al., 2010; Yang et al., 2001). Intestinal stem cells maintained functionality both in vivo and in vitro following Paneth cell ablation induced by Atoh1 deficiency (Durand et al., 2012; Kim et al., 2012). Inhibition of Atoh1 after CHIR or CHIR+VPA treatment helps maintain the self-renewal program of intestinal stem cells.

Thus, by using a combination of growth factors and small molecule inhibitors, in vitro control of the self-renewal of Lgr5 ⁺ intestinal stem cells and their differentiation into differentiated cell types in the intestinal epithelium, which closely mimics the intestinal epithelium, has now been achieved. Biology (Figures 12A and 12B). Under physiological conditions (FIG. 12A), self-renewal and differentiation of ISCs are controlled through the cooperation of Wnt and Notch pathways. Activation of both pathways (indicated by Wnt On and Notch On) maintains ISCs in an undifferentiated, self-renewing state. Inactivation of the Notch pathway (Notch Off) leads to the specification of secretory cell types, and further inactivation of the Wnt pathway (Wnt Off) leads to goblet cell differentiation. Continuous activation of the Wnt pathway in the absence of Notch leads to Paneth cell differentiation. There is no strong dependence on the Wnt pathway for enteroendocrine cell differentiation. Alternatively, sequential Notch activation and Wnt inactivation lead to intestinal epithelial cell differentiation. When Lgr5+ stem cells were cultured in vitro (Fig. 12B), CHIR99021 activated the Wnt pathway and inhibited enterocyte differentiation, but VPA alone or together with CHIR inhibited secretory cell specification. The combination of CHIR and VPA maintains ISCs in an undifferentiated, self-renewing state. Inhibition of the Notch pathway by DAPT leads to the specification of secretory cell types, and further addition of CHIR leads to Paneth cell differentiation, while addition of the Wnt pathway inhibitor IWP-2 leads to goblet cell differentiation. Alternatively, the combination of IWP-2 and VPA that induces differentiation and inhibits secretory cell specification results in intestinal epithelial cell differentiation.

Example 5. Proliferation of Lgr5-positive stem cells derived from the inner ear is increased in the presence of CHIR and VPA

The sensory hair cells of the mammalian organ Corti in the inner ear do not regenerate when damaged. Li et al., 2003 found that the mature follicle sensory epithelium contains cells displaying characteristics characteristic of stem cells. These inner ear stem cells can be cultured in vitro as suspension spheres in the presence of EGF, bFGF and IGF-1 (Li et al., 2003). It was later found that post-mitotic Sertoli cells maintain the ability to divide and trans-differentiate into new hair cells in culture (Patricia et al., 2006, Nature), suggesting that these Sertoli cells may be inner ear stem cells. Purified cochlear Sertoli cells can be cultured in vitro on embryonic periauricular mesenchymal feeder cells in the presence of EGF, bFGF (Patricia et al., 2006). Shi et al. found that a subset of Sertoli cells in the neonatal and adult murine cochlea expressed Lgr5, a marker of mature stem cells (Shi et al., 2012). Importantly, Lgr5-positive cells could be isolated and cultured in single-cell suspensions in the presence of EGF, bFGF, and IGF-1 and displayed enhanced self-renewal capacity compared to Lgr5-negative cells. In previous suspension culture methods utilized for inner ear stem cell cultures, only about 0.068% of total cells (Li et al., 2003) or 2% of sorted Lgr5-positive cells could form spheroids (Shi et al., 2012), which may It is because the cell growth environment is not suitable. As described herein, a highly efficient in vitro culture system for inner ear stem cells has now been developed.

Isolated mouse cochlea from P1 to P2 Lgr5-GFP mice contained Lgr5 positive cells, as shown in Figure 13A. The same culture conditions (EGF, noggin, R-spondin1 or "ENR") as used in Lgr5+ intestinal stem cell cultures were first established. As shown in Figure 13B, the combination of EGF, noggin and R-spondin1 increased the efficiency of colony formation from single cochlear epithelial stem cells compared to EGF alone. As expected, unlike CHIR alone, the combination of CHIR and VPA greatly increased the colony formation efficiency, cell proliferation and GFP expression of cells. Surprisingly, removal of noggin from the ENR-CV combination ("ER-CV" condition) resulted in slightly higher colony formation efficiency and higher expression levels of GFP, as shown in bright field and GFP in Figure 13B as shown in the image. These results indicated that Wnt pathway activation through R-spondin1 or CHIR promoted the proliferation of inner ear stem cells, and that CHIR and VPA greatly promoted the proliferation and self-renewal of inner ear stem cells.

Mitogenic growth factors including EGF, bFGF, and IGF-1 were previously used in suspension culture systems and were shown to promote spheroid formation of isolated inner ear stem cells (Li et al., 2003; Shi et al., 2011). The effect of CHIR and VPA in the presence of these growth factors as described in Table 1 was tested below.

Table 4. Cell Culture Solutions

Isolated Corti organs from Lgr5-GFP mice were dissociated into single cells using Accutase and cultured in various combinations of soluble factors and small molecules in Matrigel for 8 days. The resulting cultures were further dissociated into single cells and analyzed using FACS. Consistent with previous results, the addition of CHIR and VPA, rather than CHIR or VPA alone, greatly increased cell proliferation (9- to 20-fold) and GFP expression, as measured by the percentage of GFP+ cells (60-fold) and the relative GFP intensity (2x) is shown (Figures 14A and 14B). In addition, the combination of EGF, bFGF and IGF-1 (referred to as EFI) improved cell proliferation and GFP expression compared to ENR conditions (Figures 14A-14C).

To further investigate the effect of individual growth factors when combining CHIR and VPA, growth factors including mitogenic growth factors (EGF, bFGF, and IGF-1) and the Wnt agonist R-spondin1 combined with CHIR and VPA were tested. tested. Addition of EGF to CV conditions greatly increased cell proliferation, as indicated by increased cell numbers in culture. Addition of bFGF but not IGF-1 or R-spondin 1 to EGF+CV further increased cell proliferation and GFP expression (Fig. 14D). While addition of IGF-1 or R-Spondin 1 to the EGF+bFGF combination slightly increased GFP expression (Fig. 14E), the inventors found that it was not critical for maintaining proliferation and GFP expression of cultured cells (Fig. 14F).

Example 6. Lgr5 positive small intestinal stem cells form transplantable crypts

To examine the potential of transplanting intestinal stem cells, intestinal crypt engraftment in healthy colon tissue was tested in vitro. Colon tissue was collected from wild-type mice and dissected longitudinally. 1 cm segments were removed and rinsed with PBS. The epithelial layer was removed by scraping with a scalpel, and the tissue was placed in a 24-well plate. Small intestinal crypts isolated from Lgr5-GFP mice were stained with DiD membrane dye and plated on top of colon tissue in 5 μl-10 μl of crypt medium containing the following materials: Advanced DMEM/F12 (Invitrogen), 2mM GlutaMax (Invitrogen), 10mM Hepes (Invitrogen), 100U/ml penicillin/100ug/ml streptomycin (Invitrogen), 1xN2 supplement (Invitrogen), 1xB27 supplement (Invitrogen), 50ng/ ml EGF (Peprotech), 500 ng/ml R-spondin 1 (R&D Systems), 10 μΜ Y-27632 (Rho kinase inhibitor, Sigma-Aldrich) and 100 ng/ml noggin (Peprotech). The above tissues were further incubated in a humidified environment at 37°C for 30 to 60 minutes to allow the crypts to attach. Thereafter crypt medium was added to the wells and the crypts were cultured for an additional 7 days. The inoculated crypts attached to the colon and expanded within 24 hours (Figure 15). Fluorescence images showed that crypts implanted on the colon over 48 hours (Figure 16) and maintained Lgr5-GFP expression for at least 1 week (Figure 17).

To further test the engraftment ability of small intestinal crypts, a TRUC mouse model exhibiting spontaneous ulcerative colitis and mimicking human conditions was used. Prolapsed tissue was excised from TRUC mice and rinsed with PbS and placed in 24-well plates. Small intestinal crypts were stained with DiD and placed on top of the prolapsed tissue. The tissue is then incubated in a humidified environment at 37°C for 30 minutes to 60 minutes to allow crypts to attach. The prolapsed tissue and crypts were further cultured in vitro for 2 days. As expected, the crypts were implanted on the prolapsed tissue (Figure 17).

Example 7. The patch culture system of small intestinal organoids simulates a three-dimensional physiological environment

In vitro culture systems capable of supporting the growth of large-scale organized three-dimensional cellular structures (eg, organoids) on submucosal scaffolds have been developed.

An improved small intestinal submucosa ("SIS")-based culture system for three-dimensional tissue constructs was prepared by seeding the submucosa with preselected cell types and promoting growth with a unique collagen-like coating, as described below . The coating was initially a viscous liquid pre-polymer that was used to cover seeded early cells or organoids (subcultured from cells), as well as the SIS substrate to encase the cells in the collagen residue (Figure 19E and 19F). After polymerization, the liquid solidifies to maintain its position in contact with the cell membrane and SIS and to facilitate organoid expansion. It has now been found that altering the composition of the SIS and this coating promotes cell attachment and growth. This promotes tissue maturation in vitro as opposed to in vivo. This is a unique improvement over other similar submucosa-based synthetic systems, as three-dimensional expansion of adherent cells into large endogenous organoids is achieved prior to transplantation.

In addition, methods have also been found to support the growth of three-dimensional organoids on submucosa at a rate comparable to Matrigel without using a gel layer. The system consists of a vertebrate SIS and preselected cells seeded on the SIS patch. Prior to cell seeding, preselected bioactive agents were infused into the patch to support the gel-free culture system (Figures 19C and 19D).

To develop the patch culture system, various combinations of SIS substrates and collagen overlays with infused growth factors were investigated (Figures 19E and 19F). This enables the creation of more physiological tissue interfaces with a transition from firm (SiS) to soft (collagen) matrices. It was determined that seeded cells and organoids coated with collagen residues provided a similar three-dimensional environment to that provided by Matrigel. Therefore, this system is a suitable replacement for Matrigel in the culture of three-dimensional organoid constructs. Most of the seeded cells or organoids were both attached to the SIS on the lower half of the cell membrane and surrounded by polymerized collagen on the unattached regions of the membrane (FIG. 19E, inset). Thus, each cell membrane is functionally encapsulated in some form of matrix (SIS or collagen). In some samples, various bioactive agents were employed in addition to SIS alone to support cell and organoid seeding, growth and differentiation (Fig. 19F). Although applicants describe focusing on biomolecules specific to small intestinal stem cell cultures, it is claimed that biomolecules can be tailored to assist the growth of other seeded cells from different tissues including pancreas, breast, liver and stomach tissues. Thus, tissue-specific biomolecules may be selected from the group consisting of: antivirals, antimicrobials, antibiotics, amino acids, peptides, proteins, glycoproteins, lipoproteins, antibodies, steroids, antibiotics, antifungals, cytokines , vitamins, sugars, lipids, extracellular matrix, extracellular matrix components, chemotherapeutic agents, cytotoxic agents, growth factors, anti-rejection agents, analgesics, anti-inflammatory agents, viral vectors, protein synthesis cofactors, hormones , endocrine tissue, synthetics, enzymes, polymer-cell scaffolds with parenchymal cells, angiogenic drugs, small molecules, nanoparticles, collagen lattices, antigenic agents, cytoscaffolds, nucleic acids, cell attractants.

Initially, crypts were isolated according to previously described methods (Sato et al., 2009, Yui et al., 2012). The murine small intestine was isolated, dissected longitudinally and flushed with ice-cold PBS to remove luminal contents. Fragments were cut into 2mm pieces, transferred to a 50ml falcon tube and washed gently in 50ml ice-cold PBS with a 10ml pipette. The supernatant was removed and the process continued until the supernatant had cleared. The fragments were incubated in PBS containing 2 mM EDTA for 45 minutes at 4°C to release the crypts. The supernatant was removed and the fragments were pipetted off with 50ml PBS. Once the supernatant was confirmed to contain crypt components, the suspension was filtered through a 70 µm cell strainer and spun in a centrifuge at 300 g for 5 min. Crypts were resuspended in 10 ml ice-cold basal medium (containing Advanced DMEM/F12 (Invitrogen), 2 mM GlutaMax (Invitrogen), 10 mM Hepes (Invitrogen), and 100 U/ml penicillin/100 μg/ml streptomycin (Invitrogen)) and Transfer to a 15ml centrifuge tube. PBS washes were repeated and crypts were spun at 200g for 2 minutes to remove single cells. Crypts were counted and plated at a concentration of 1000 crypts/well onto matrigel or collagen I (made from 100 μl 10x PBS, 4.9 μl NaOH, 684 μl HO, and 211 μl type I collagen (rat tail high concentration 9.49 mg/ml ; BD Biosciences) in a 48-well plate, each well contained 200 μl of matrix. After polymerization of selected gel products, add 500 μl 1x standard crypt medium (serum-free) containing advanced DMEM/F12 (Invitrogen), 2mM GlutaMax (Invitrogen), 10mM Hepes (Invitrogen), 100U/ml penicillin /100 μg/ml Streptomycin (Invitrogen), 1x N2 Supplement (Invitrogen), 1x B27 Supplement (Invitrogen), 50ng/ml EGF (Peprotech), 500ng/ml R-spondin 1 (R&D Systems), 10 μM Y-27632 (Rho kinase inhibitor, Sigma-Aldrich) and 100 ng/ml noggin (Peprotech). Cells were grown for 4 to 5 days before seeding onto the patches, with media changes every other day. Y-27632 was included in the medium only for the first 48 hours.

After 4 to 5 days in culture, Lgr5+ organoids were passaged using a modification of the previously described strategy (Sato et al., 2009). Media was removed from Matrigel, then manually disrupted with a p1000 pipette and transferred to BSA-coated 15ml centrifuge tubes. Collagen gels were incubated in DMEM containing collagenase type XI for 5 min at 37°C and then transferred to BSA-coated 15 ml centrifuge tubes. Add basal medium and gently agitate the organoids by checking frequently with an inverted microscope until most of the organoids are single crypts. The organoids were rinsed in 10 ml basal medium and centrifuged at 200 g for 2 minutes. Resuspend the pellet in crypt medium at a concentration of 500 single-crypt organoids/500 μl.

Patches were generated and prepared for seeding inside the wells of a standard 48-well plate (1 patch/well, chamber side up). The SIS was cut to the desired length to cover the bottom of each well (approximately 1 cm for a 48-well plate). SIS was isolated as previously described (Badylak et al., 1989). Using blunt-ended forceps, transfer each SIS fragment to the bottom of the well and unfold carefully to its full diameter, luminal side up. Localization was confirmed by analysis under an inverted microscope to visualize crypt acellular remnants on superficial surfaces.

Depending on the desired conformability and strength, multiple layers of SIS can be layered and combined. In this case, each fragment can be spread on top of the other to achieve the desired number of fragments, and the patch is lightly pressed with tweezers and allowed to air dry at 5% _CO2 , 37°C for 5 minutes. Prior to inoculation, each patch segment was dehydrated by passive evaporation for 24 hours and concentrated crypt medium and optionally small molecules as described below. Specifically, each segment of the patch was placed and spread in a well of a 48-well plate (cavity side up), and 100 μl of concentrated factors (EGF, noggin, R-spondin 1, Y-27632, Valproic acid and CHIR) were incubated at 37°C in 5% CO ₂ for 24 hours.

Individual 500 μl samples of single-crypt organoids were deposited into wells containing the patch substrate and incubated at 5% CO ₂ and 37° C. for 24 hours ( FIG. 20A ). Inoculated patches were maintained in medium for 24 hours to allow for firm attachment and trophic support from growth factors embedded in the patch (Figure 20B).

In some instances, a thin collagen gel residue (referred to as a gel-patch) is coated on top of the patch/organoid complex to provide each organoid with a minimal but functional three-dimensional surroundings. Physical and chemical cues obtained from the cell surface enhance proliferation of three-dimensional cellular structures in order to replicate physiological morphology (Seidi, A. et al., 2011).

A layer of collagen I matrix (20 μl-40 μl) was spread on the inoculated patch, responsible for preventing the gel from spreading beyond the patch by means of surface tension ( FIG. 20C ) and the well plate was incubated at 5% CO ₂ , 37° C. for 30 minutes. education. Crypt medium (500 μl) was deposited into each well and changed every other day.

In some instances, the patches were incubated in growth factors prior to seeding to check whether they facilitated the attachment of the organoids in the first 24 hours. Therefore, GF-perfused (including EGF, noggin, R-spondin 1, Y-27632, valproic acid, and CHIR) and non-perfused patches (SIS in PBS) were inoculated. In this test, non-perfused patches were replaced with basal medium so that the growth factors of the medium were also not available to the organoids.

Growth of small intestinal organoids was assessed by quantifying the number of crypts per organoid in seven separate systems: with perfused growth factors (referred to herein as GF, and including EGF, noggin, R-spondin 1. Matrigel (control) of Y-27632, valproic acid and CHIR), bare patch with perfused GF but no collagen overlay, collagen I gel only, with GF added directly to culture medium ( Collagen I gel including EGF, noggin, R-spondin 1, Y-27632, valproic acid and CHIR), GF with embedded gel itself (including EGF, noggin, R-spondin1, Y- 27632, valproic acid and CHIR) and bare patches of GF without collagen coating or perfusion. All media between the systems were standard, replaced every 1 day, and included EGF, noggin, R-spondin, except the collagen I group with GF and small molecules added directly to the media 1. Y-27632 (only the first 48 hours). Standard crypt medium is as described above.

Experiments were performed over 96 hours and daily quantification of organoid growth was documented by visual inspection of the number of crypts per organoid. The gel-patch system with GF was able to support organoid growth at levels comparable to the Matrigel control (Figure 19). Bare patches without GF cannot support measurable organoid growth. On closer inspection, bare SIS patches appeared to grow Lgr5+ cells in sheets rather than three-dimensional organoids.

However, bare patches with perfused GFs (EGF, noggin, R-spondin 1, Y-27632, valproic acid, and CHIR) supported organoid growth at the same level as gel-patch systems and Matrigel . This suggests that the gel-free culture system can sustain short-term 3D organoid growth at the same level as Matrigel with sufficient GF support. While collagen I by itself contributes to moderate organoid growth, SIS infused with GF is a beneficial surrogate for the three-dimensional growth-promoting effect of collagen. Furthermore, when the same GFs (EGF, noggin, R-spondin 1, Y-27632, valproic acid, and CHIR) were directly added to the medium of collagen I gel cultures, organoid growth was maintained at low s speed. In addition, organoid growth was maintained at a lower rate when collagen I gels were prepared with the aforementioned GF directly embedded in the gel prior to seeding. GFP signal was maintained throughout the gel-patch system (representative examples in Figures 21B and 21C). The observation that there is no support for structured organoid growth on bare patches (SIS without collagen coating or GF) confirms the importance of adequate physical and chemical cues to facilitate three-dimensional structure.

Only SIS or collagen have been used in the literature as base scaffolds for cell seeding, resulting in the formation of cell monolayers (Baumert et al., 2007; Campodonico et al., 2004; Feil, G. et al., 2006; Zhang, Y. et al., 2000 ). In contrast, growing cells at the interface of these two matrices favors 3D organoid growth compared to monolayer growth. This more closely mimics the physiological environment, enabling accelerated and structured growth. Importantly, these results describe a patch culture system for small intestinal organoids as an excellent alternative to Matrigel. Matrigel-based grafts in animal models have encountered significant obstacles in their progress to human models, the most critical of which include biocompatibility issues. Growing three-dimensional cell-based structures often requires embedding thick matrix gels. The patch culture system described above overcomes this need while providing comparable results. Replacing Matrigel with a combination of endogenous extracellular matrix materials and specific bioactive growth factors circumvented biocompatibility issues while maintaining ex vivo growth of 3D organoids. Time-lapse images of three-dimensional ex vivo organoid expansion from the initial species are shown in Figure 22.

It was assessed whether incubation of patches in growth factors prior to seeding facilitated organoid attachment during the first 24 hours. The seeding efficiency of growth factor-infused patches (including EGF, noggin, R-spondin 1, Y-27632, valproic acid, and CHIR) was compared with untreated patches (stored in PBS). The test was performed by determining the percentage of organoids remaining after medium flushing at 4 hours and 12 hours when cells were cultured alone in medium lacking growth factors (basal medium only). When SIS was omitted and organoids were seeded directly onto plastic collagen-coated and non-collagen-coated wells, all organoids dissociated within 24 hours. However, the SIS patch maintained most of the organoids at 24 hours, both in structure and GFP expression. Improved attachment was observed when cells were seeded onto patches infused with growth factors, including EGF, Noggin, R-spondin 1, Y-27632, Valproic acid and CHIR. Thus, growth factor perfusion can also be used to provide appropriate nutrients and factors during culture and subsequent transplantation, by bridging the gap with cell engraftment.

Example 8. Implanted patches exhibit growth promoting properties in vivo

An acellular, gel-free variant of the patch system was tested to assess the mucosal healing properties in vivo. A rat surgical model of mucosal defects was designed in order to test the growth promoting properties of the implanted patches in vivo. The implant patch was assembled by carefully spreading a portion of the SIS (luminal side up) on a 6 mm circular poly(ethylene sebacate)urethane (PGSU) backing. The patches were incubated in 5% CO2, 37°C for 30 minutes to allow PGSU and SIS to bind. A 4 mm defect was created in the stomach wall by punch biopsy, as shown in Figure 23. An acellular patch (6 mm in diameter) was placed on the external gastric wall, carefully covering the defect with the material of choice. By the modified Graham patch method ( et al., 1996) secure the patch using sutures and nearby connective tissue. Three cell-free patch variants were applied including: a) PGSU-backed SIS patch (no GF); b) perfused GF (EGF, Noggin, R-spondin 1, Y-27632, PGSU-backed SIS patch with valproic acid and CHIR); and c) PGSU-backed only (no SIS). Peritonitis was not observed in any rat at any time point. One week after implantation on the mechanically induced gastric wall defect, the gastric tissue containing the defect and the patch implant was harvested for histological examination of the tissue.

It was hypothesized that the implanted patch variants would show varying degrees of mucosal healing. Gross inspection revealed a significant benefit of SIS patches with GF, as defects were epithelialized and closed. Partial occlusion without epithelialization was observed in SIS patches without GF, while no occlusion or epithelialization was observed in PGSU only (control) patches. Histological examination revealed mild inflammation in both SIS patches with and without GF, but no leakage of gastric contents. Histological examination of the PGSU-only patch revealed moderate inflammation and the presence of giant cells, possibly in response to leakage of gastric contents. Thus, the patch culture system described herein can be directly transplanted from a dish to a patient with increased translational potential since the patch is rigid but less easily placed on small surfaces due to its lower height profile. Obstruction within a space (eg, small bowel lumen, vascular space).

Example 9. Culture of Human Small Intestinal Crypt/Stem Cells

Human intestinal crypts were isolated from resected normal intestinal specimens and cultured as described in Example 1. The same cell culture solution (comprising CHIR99021 combined with VPA or Tubastatin A added to ENR (EGF, Noggin, R-Spondin 1) conditions) as used in mouse intestinal stem cell/crypt culture was mixed with Published human intestinal stem cells/crypts were compared with cell culture solutions (Jung et al., 2011; Sato et al., 2011). RT-PCR was used to assess the maintenance of epithelial stem cells in culture, specifically by determining self-renewal or differentiation status. LGR5 was used as a stem cell marker, and ALP1, MUC2, CHGA, and LYZ were used as differentiation markers. Cell growth was assessed by counting the number of cells in the culture and by observing the morphology and size of the colonies.

Similar to mouse intestinal stem cell cultures, the combination of CHIR+VPA or CHIR+tubastatin A greatly promoted the expression of the stem cell marker LGR5, indicating that the cultured cells were enriched for stem cells (Figure 24). In particular, culture conditions comprising CHIR and VPA or CHIR and tubastatin A performed better than published conditions in promoting LGR5 expression (Figure 24). Additionally, individual components that showed improvements to the medium including A83-01 (ALK4, 5, 7, Tgf-β inhibitor), SB202190 (p38 inhibitor), and nicotinamide (vitamin B derivative) were tested. tested. It was determined that 10 mM nicotinamide, when added to CHIR+VPA conditions, increased the proliferation of human intestinal crypts, as indicated by increased cell numbers in culture (Figure 25A), without a major effect on LGR5 expression (Figure 25B) . While the combination of A83-01 and SB202190(AS) increased cell proliferation (Fig. 25A), they dramatically decreased LGR5 expression (Fig. 25B). In addition, lower concentrations of VPA (0.5 mM compared to that used in mouse cultures (1 mM-2 mM)) increased cell proliferation in human intestinal crypts (Fig. 25A). In summary, it was determined that the culture conditions containing EGF, noggin, R-spondin 1, CHIR, VPA (0.5 mM) and nicotinamide or EX527 are the optimal culture conditions for human small intestinal stem cells. Under these conditions, isolated intestinal crypts grew into colonies comparable to mouse intestinal stem cells (Fig. 26).

Example 10.

To test the in vivo effects of CHIR and VPA on intestinal epithelial cells, CHIR99021 (30 mg/Kg in 100 μl DMSO) and VPA (200 mg/Kg in 100 μl water) were administered by gavage to 4- to 6-week-old female Lgr5-GFP pups. mouse. Control mice were given a mixture of 100 μl DMSO and 100 μl water. Drugs were administered every 48 hours for 7 days (Day 0, Day 2, Day 4 and Day 6). On day 7, mice were sacrificed and small intestine tissues were collected. Small intestines were further rinsed with PBS, fixed with 4% PFA for 12 hours, embedded in paraffin and stained using standard hematoxylin and eosin (H&E) staining strategy. Images were collected using an inverted microscope (EVOS, Advanced Microscopy Group). In vivo administration of CHIR and VPA increased crypt size after 3 administrations over a 7 day period (Figure 27).

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All patents, patent applications, and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

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other implementations

It is to be understood that while the foregoing description has been described in conjunction with the invention, that the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims (10)

1. a kind of cell culture solution, it includes the inhibition of the inhibitor of bone morphogenetic protein, GSK-3 β Agent, the reagent and histone deacetylase inhibitors combined with g protein coupled receptor containing full asphalt mixture 5.

2. a kind of cell culture solution, it includes the inhibitor of bone morphogenetic protein, at least about 3 μM of CHIR99021 and group egg White deacetylase inhibitors.

3. cell culture solution as claimed in claim 1 or 2, wherein the histone deacetylase inhibitors are Pan- Hdac inhibitor.

4. cell culture solution as claimed in claim 3, wherein the Pan-HDAC inhibitor is selected from mould by valproic acid, bent Gu The group of plain A, Vorinostat and suberoyl hydroxamic acid (SBHA) composition.

5. cell culture solution as claimed in claim 1 or 2, wherein the histone deacetylase inhibitors are HDAC6 Inhibitor.

6. cell culture solution as claimed in claim 5, wherein the HDAC6 inhibitor is selected from by native Ba Xing, soil bar statin A The group formed with compound 7.

7. cell culture solution as claimed in claim 1 or 2, wherein the inhibitor of the bone morphogenetic protein is selected from by head Element occurs, element, follistatin, DAN, the albumen of the knob domain of cysteine containing DAN, osteosclerosis albumen, primitive gut shape occur for notochord At albumen, uterine sensibility related gene -1, Connective Tissue Growth Factor, inhibin, BMP-3 and Dorsomorphin composition Group.

8. cell culture solution as described in claim 1, wherein the inhibitor of the GSK-3 β be selected from by CHIR99021, LiCl, BIO- acetoxime, CHIR98014, SB 216763, SB 415286,3F8, willing Paro ketone, 1- azepine are agreed Paro ketone, TC-G 24, TCS 2002, AR-A 014418, TCS 21311, TWS 119, BIO- acetoxime, 10Z- Haimen Plug, GSK-3 beta inhibitor II, GSK-3 beta inhibitor I, GSK-3 beta inhibitor XXVII, GSK-3 beta inhibitor XXVI, FRATtide The group of peptide, Cdk1/5 inhibitor and Bikinin composition.

9. cell culture solution as described in claim 1, wherein described and 5 knot of g protein coupled receptor containing full asphalt mixture The reagent of conjunction is selected from the group being made of R-spondin 1, R-spondin 2, R-spondin 3 and R-spondin 4.

10. cell culture solution as described in claim 1, wherein the inhibitor of the GSK-3 β be selected from by The group of CHIR99021 composition, the reagent combined with g protein coupled receptor containing full asphalt mixture 5 is R-spondin 1, And hdac inhibitor is valproic acid.